Mini-Review: Recent Technologies of Electrode and System in the Enzymatic Biofuel Cell (EBFC)

: Enzymatic biofuel cells (EBFCs) is one of the branches of fuel cells that can provide high potential for various applications. However, EBFC has challenges in improving the performance power output. Exploring electrode materials is one way to increase enzyme utilization and lead to a high conversion rate so that efﬁcient enzyme loading on the electrode surface can function correctly. This paper brieﬂy presents recent technologies developed to improve bio-catalytic properties, biocompatibility, biodegradability, implantability, and mechanical ﬂexibility in EBFCs. Among the combinations of materials that can be studied and are interesting because of their properties, there are various nanoparticles, carbon-based materials, and conductive polymers; all three have the advantages of chemical stability and enhanced electron transfer. The methods to immobilize enzymes, and support and substrate issues are also covered in this paper. In addition, the EBFC system is also explored and developed as suitable for applications such as self-pumping and microﬂuidic EBFC.


Introduction
Enzymatic biofuel cells (EBFCs) is one of the branches of biofuel cells, other than microbial fuel cells (MFCs), that constitutes one of the emerging areas of green energy and nanotechnology [1] to reduce carbon dioxide emissions [2]. EBFC is easily applied and provides power [3] in implantable medical devices such as cardiac pacemakers [4], drug delivery [5], implantable artificial organs [6], and wastewater treatment [7]. The EBFC is also used as a biosensor for biomolecules detection, such as toxic pollutants [8] and glucose detection [9]. Detection of toxic pollutants can protect the environment from being exposed to the danger of molecule release at low concentrations [10]. In addition, the use of EBFC in medical devices is seen to bring advantages due to the small size of the EBFC [11], its flexibility, light weight, and easy portability [12], and more importantly, its sufficient usage at low power [13]. Making EBFCs for various uses is simple and low cost compared to other types of fuel cells. Each fuel cell has a lifetime and has its way of disposal. In contrast, EBFC, which has a small size, is accessible to disposal [14]. The EBFC operating process is also easy, rapid to start-up, eco-friendly, has an anti-interference performance [3], and is easy to operate at room temperature and neutral pH [15,16].
Despite having many advantages, EBFC also has its challenges, where there is still room for improvement to increase the potential usage of EBFC. Combining various substances with enzymes carries the challenges of biocatalytic properties, biocompatibility, biodegradability, implantability, and mechanical flexibility in EBFC [17]. The shortcoming in the EBFC system leads to integrated miniaturization issues [18], lower power density, poor operational stability, lower voltage output [19], lower energy density, inadequate durability, instability in long-term application [15,20], and incomplete oxidation of fuel [21].
Various non-toxic chemical fuels are used in EBFC, such as glucose, lactate, urate, alcohol, amines, starch, and fructose [22], and have a specific enzyme to oxide these fuels; for example, the cellobiose dehydrogenase is used to oxide the lactose in the EBFC [23]. However, glucose is widely used because it has the advantages of being abundant, easy to obtain, cheap [24], and involved in various functions [25]. An enzyme is used in EBFC as a catalyst. In the anode, the enzyme commonly oxidizes the glucose into gluconolactone and produces electrons and protons. Glucose oxidase is widely used because it has advantages such as availability [26], high selectivity [27], and high catalytic activity to oxide glucose [25], self-discharge, low mix potential [28], and ease of use in a membrane-less EBFC system. At the same time, the enzyme at the cathode will complete the reduction reaction by using electrons and protons from the anode and then generate electricity. The working principle in EFC at the anode and cathode is given below: Complete oxidation of glucose: Anodic glucose oxidation (GOx): Cathodic oxygen reduction (laccase):  Figure 1. Enzymes immobilized on an electrode surface via (a) physical adsorption to a polymer, (b) covalent attachme to a polymer (as shown by the black and white tethers), or (c) encapsulation in [35]. Copyright 2012 Elsevier.
A convenient immobilization method recommended for interaction between e and support [30] needs to increase the enzyme's storage, solvent, pH, and thermal s [3,33,38]. However, some factors have been detected behind enzyme-support inter that lead to low power and energy density. There is the inadequate and inefficien action [16] as well as imperfect [20] and low enzyme loading between enzyme and s that reduces electron transfer [39], leaches out enzyme [26], leads to degradation i term operation, causes potential enzyme poisoning when using non-biocompatibili port materials, reduced lifetime [21], and limited enzyme stability [24,39]. Variou rials and methods are used to alleviate this problem that give different chemical, m ical, and electrical properties [39,40]; for example, Ji et al. [41] reduced the leach enzyme problem by linking the interaction between hemin and carbon nanotubes t functionalized amine groups to form amide bonds, as shown in Figure 2. Meanwhi conjugated the GOx enzyme with two poly (dimethyl-diallyl ammonium chlorid ducing a sandwich structure, which maintains GOx loading. The stability of this ch bonding has kept 82.1% activity for four weeks. Figure 2 also shows that various i tion experiments produce different electrochemical reaction activities. In addition valent bonds formed through the amino group, crosslinking agents are also p through carboxyl (-COOH) groups [42]. Ji et al. [43] produced polyethyleneimine ( which the redox polymer is used to entrap and bind GOx enzyme with F-N/CNT a thus making physical interaction through electrostatic attraction. PEI offers advant increasing electron transfer because it has a lower glass transition that mobilizes b the polymer structures and has high stability, sensitivity [42], and conductivity addition, US et al. [18] minimized enzyme leaching by producing laser-induced gr with CNTs in microfluidic EBFCs. The high porosity and low cost of this material in the active site for the enzyme and give 1.37 times higher power density than w CNTs. Shakeel et al. [1] produced carbon composites by combining MWCNT with (non-perfluorinated sulfonated penta block copolymer) to form an excellent ma Shakeel 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. Kraton has high ion mobility, film-forming ability, and high proton conductivity that can produce a current as high as 1.14 mA/cm 2 at a glucose concentration of 60 mM. Kang et al. [39] developed a rectangular carbon tube with a concave surface to immobilize the GOx and laccase (LAC) from rectangular polypyrrole (RPPy). RPPy is carbonized at high temperatures so that the resulting surface structure has defects at a high degree of disorder and low degree of intralayer binding of the amorphous carbon. In addition, Arjun et al. [2] used pyrene carboxylic acid with MWCNT and reduced graphene oxide-ceria for the electrostatic immobilization of enzymes via calcium ions. The calcium ion is produced on the electrode surface by dipping the electrode in a calcium chloride solution for two hours.
The electrode consists of a substrate and support material. The support material is used to support the enzyme and will be on the surface of the substrate. The commonly used substrate is carbon cloth [44], carbon paper [45], indium tin oxide (ITO) [22], glassy carbon electrode [46], and buckypaper [47], because these carbon-based materials provide advantages such as high compatibility, being user-friendly, and being environmentally friendly and made of reusable material [40], through physical adsorption or chemical bonding to form the carbon composites [39]. Substrates are rarely studied in EBFCs, but Shen et al. [48], using graphene paper [49], used the reduction method followed by crosslinking to strengthen the mechanical stability of the material and used it for the anode and cathode in the EBFC, as shown in Figure 3. In the EBFC system developed, authors use pyrroloquinoline-dependent glucose dehydrogenation (PGG-GDH) and bilirubin oxidase (BOx) as the enzyme anode and cathode. Wan et al. [50] used carbon paper to solve the problem of the low solubility of the gas molecule in the aqueous solution with polytetrafluoroethylene to increase its hydrophobicity. Low power due to low oxygen solubility was increased by producing power of 53.0 µW/cm 2 at 0.45 V. Rewatkar et al. [51] applied the buckypaper as a bioelectrode for both GOx and laccase at the bioanode and biocathode without a redox co-factor, which offers various advantages, such as highly efficient electron transfer, scalable production, high electrical conductivity, and large specific areas [52], respectively. Niiyama et al. [44] modified the carbon cloth with MgO-template porous carbon to further increase the electrochemically active surface area on the electrode surfaces. Both modified electrodes use an anodic and cathode binder, namely poly(vinylidenedifluoride) and polytetrafluoroethylene. The authors modified the carbon cloth using an MgO template to form mesoporous carbon and were able to control the pore size distribution to immobilize the enzyme. The electrode consists of a substrate and support material. The support material is used to support the enzyme and will be on the surface of the substrate. The commonly used substrate is carbon cloth [44], carbon paper [45], indium tin oxide (ITO) [22], glassy carbon electrode [46], and buckypaper [47], because these carbon-based materials provide advantages such as high compatibility, being user-friendly, and being environmentally friendly and made of reusable material [40], through physical adsorption or chemical bonding to form the carbon composites [39]. Substrates are rarely studied in EBFCs, but Shen et al. [48], using graphene paper [49], used the reduction method followed by crosslinking to strengthen the mechanical stability of the material and used it for the anode and cathode in the EBFC, as shown in Figure 3. In the EBFC system developed, authors use pyrroloquinoline-dependent glucose dehydrogenation (PGG-GDH) and bilirubin oxidase (BOx) as the enzyme anode and cathode. Wan et al. [50] used carbon paper to solve the problem of the low solubility of the gas molecule in the aqueous solution with polytetrafluoroethylene to increase its hydrophobicity. Low power due to low oxygen solubility was increased by producing power of 53.0 µW/cm 2 at 0.45 V. Rewatkar et al. [51] applied the buckypaper as a bioelectrode for both GOx and laccase at the bioanode and biocathode without a redox co-factor, which offers various advantages, such as highly efficient electron transfer, scalable production, high electrical conductivity, and large specific areas [52], respectively. Niiyama et al. [44] modified the carbon cloth with MgO-template porous carbon to further increase the electrochemically active surface area on the electrode  [41]. Copyright 2020 Elsevier.
The enzyme will attach to the surface of the support material, where the oxidation and decomposition reactions take place to produce current or power. However, it should be noted that enzymes will oxidize and reduce, such as in Equations (2) and (3), and the resulting electrons need to flow to the electrode. There are two ways of electron transfer in EBFC, namely mediated electron transfer (MET) and direct electron transfer (DET) [22]. The redox mediator is used in MET as an electron acceptor for electron transfer. These mediators are encouraged to attach at the electrode [28] to increase electron transfer by covalently attaching to a polymer backbone or directly adsorbing on the electrode surface by weak noncovalent immobilization or physical adsorption by drop-casting; both methods still carry the problem of mediator leaching. Tsuruoka et al. [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 redoxmediating 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, 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]. Korkut et al. [55] synthesized a mediatormodified 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 enzyme will attach to the surface of the support material, where the oxidation and decomposition reactions take place to produce current or power. However, it should be noted that enzymes will oxidize and reduce, such as in Equations (2) and (3), and the resulting electrons need to flow to the electrode. There are two ways of electron transfer in EBFC, namely mediated electron transfer (MET) and direct electron transfer (DET) [22]. The redox mediator is used in MET as an electron acceptor for electron transfer. These mediators are encouraged to attach at the electrode [28] to increase electron transfer by covalently attaching to a polymer backbone or directly adsorbing on the electrode surface by weak noncovalent immobilization or physical adsorption by drop-casting; both methods still carry the problem of mediator leaching. Tsuruoka et al. [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, 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 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]. Herkendell 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. Herkendell 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 Appl. Sci. 2021, 11, 5197 6 of 27 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]. 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. Herkendell 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 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. The essential criteria for electrode development are a robust adsorption site [14] for enzyme and mediator interaction on the support and support-substrate interaction. The result of a strong interaction between enzyme and support, mediator and support, and support and substrate reduces overpotential, and gives high durability and stability in long-term operation.
The development of suitable and robust interaction on the support material is needed to reduce the problems encountered in the enzyme immobilization process. Good support material with a high surface area and low aggregation can provide many sites for enzyme 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. The essential criteria for electrode development are a robust adsorption site [14] for enzyme and mediator interaction on the support and support-substrate interaction. The result of a strong interaction between enzyme and support, mediator and support, and support and substrate reduces overpotential, and gives high durability and stability in long-term operation.
The development of suitable and robust interaction on the support material is needed to reduce the problems encountered in the enzyme immobilization process. Good support material with a high surface area and low aggregation can provide many sites for enzyme immobilization. Not only that, the mesoporous support material will help mass transport to the enzyme and increase the electronic double layer. Producing mesostructured support material needs to look at controlling uniform pore structure and pore size because the random size will cause underutilization of the support materials [60]. Development of new support materials needs to explore some of these factors to improve the ability to use supports in EBFC, namely electron transfer rate resulting from enzyme-support/electrode interaction [12], enzyme thickness, enzyme loading [15], enzyme conformation, and interfacial stability of the enzyme-support/electrode [3].

Current Development on the Bioanode and Biocathode in EBFC
A strong interaction between support materials and enzymes is needed to strengthen the enzyme's immobilization and reduce enzyme leaching. A study from Miki et al. [32] showed that the resulting covalent bond of a nitrophenyl group on the surface of a modified graphene support strengthens interaction with enzyme, which helps the DET process between the graphene support and enzyme. Meanwhile, the study from Innamuddin et al. [61] modified the CNT surface by functionalizing it with polyindole and ZnO to form an interaction support with enzyme and ferritin as a mediator. A high current density of 4.9 mA/cm 2 produced after consuming 50 mM glucose concentration proves an electron transfer that applies efficiently. The same group's research using nickel oxide and silver nanowires showed a higher current density of 5.4 [16] and 19.9 mA/cm 2 [56]. Investigations that use CNT as support material have been carried out widely in EBFC. For example, Sakamoto et al. [62] investigated pyrene(NHS) and Ni 2+ -NTA as a binder of the enzyme on the CNT surface for the bioanode and biocathode. Kang et al. [39] developed a carbon tube by carbonizing rectangular polypyrrole for both the electrode bioanode and biocathode. Temperature changes cause the rectangular structure to change slightly, as shown in Figure 5. The unique morphology of the carbon tube offers a high power density of 0.350 mW/cm 2 and 0.265 mW/mg (GOx). An exciting study from Tominaga et al. [14] used MWCNT as a support and cellulose nanofiber sheet as the electrode in EBFC. After combining the bioanode and biocathode, the very thin electrode makes it easier to dispose of but gives a high power, as high as 27 µW/cm 2 . the enzyme's immobilization and reduce enzyme leaching. A study from Miki et al. [32] showed that the resulting covalent bond of a nitrophenyl group on the surface of a modified graphene support strengthens interaction with enzyme, which helps the DET process between the graphene support and enzyme. Meanwhile, the study from Innamuddin et al. [61] modified the CNT surface by functionalizing it with polyindole and ZnO to form an interaction support with enzyme and ferritin as a mediator. A high current density of 4.9 mA/cm 2 produced after consuming 50 mM glucose concentration proves an electron transfer that applies efficiently. The same group's research using nickel oxide and silver nanowires showed a higher current density of 5.4 [16] and 19.9 mA/cm 2 [56]. Investigations that use CNT as support material have been carried out widely in EBFC. For example, Sakamoto et al. [62] investigated pyrene(NHS) and Ni 2+-NTA as a binder of the enzyme on the CNT surface for the bioanode and biocathode. Kang et al. [39] developed a carbon tube by carbonizing rectangular polypyrrole for both the electrode bioanode and biocathode. Temperature changes cause the rectangular structure to change slightly, as shown in Figure 5. The unique morphology of the carbon tube offers a high power density of 0.350 mW/cm 2 and 0.265 mW/mg (GOx). An exciting study from Tominaga et al. [14] used MWCNT as a support and cellulose nanofiber sheet as the electrode in EBFC. After combining the bioanode and biocathode, the very thin electrode makes it easier to dispose of but gives a high power, as high as 27 µW/cm 2 . To reduce the leaching and optimize GOx on the electrode surface, Hyun et al. [28] developed the crosslink with chitosan and genipin as support materials, and with nitrobenzoic acid as a new mediator attached to the electrode by chemical bonding. The authors also modified the surface chitosan by distributing the abundant functional evenly on the chitosan surface structure to help covalently bond the GOx and mediator. Analysis To reduce the leaching and optimize GOx on the electrode surface, Hyun et al. [28] developed the crosslink with chitosan and genipin as support materials, and with nitrobenzoic acid as a new mediator attached to the electrode by chemical bonding. The authors also modified the surface chitosan by distributing the abundant functional evenly on the chitosan surface structure to help covalently bond the GOx and mediator. Analysis showed that the enzyme loading on the electrode surface reached 17.8 mg/mL and produced a current as high as 331 µA/cm 2 at 0.3 V vs. Ag/AgCl and low onset potential (0.05 V vs. Ag/AgCl). Duong et al. [16] directly modified the carbon cloth (CC) substrate with chitosan, commonly used as a biosensor and in biomedical applications, to support glucose oxidase enzyme. The additive assists electron transfer at the electrode, and the modification of the chitosan over the CC requires appropriate additive testing. In this study, sodium tripolyphosphate (TPP) and Nafion were selected as additives, but the results show good electron transfer using TPP. The biocompatibility of the genipin used in EBFCs is because it has lower cytotoxicity and is highly efficient to crosslink between chitosan, glucose oxidase, and the amine-containing osmium redox complex developed by Conghaile et al. [30]. The authors also increased the current density by adding multi-walled carbon nanotubes on the electrode as high as 4.9 mA/cm 2 , higher than the previous, which produced only 440 µA/cm 2 .
The Duong research group [63] further investigated the electrode's scaffold structure by determining the microstructure, electrochemical property, and enzyme loading. The interplay of the electrodes consists of chitosan, Nafion, and/or tripolyphosphate. The electrode comprising all chitosan, Nafion, and tripolyphosphate produced a high power of 1.077 mW/cm 2 , compared with the electrode containing chitosan/tripolyphosphate and chitosan/Nafion. Another study used chitosan as the composite electrode, synthesized by Sufiaul Haque et al. [64] to increase porosity and surface area for highly optimized enzyme loading. The condition of glucose at 20 mM produced a current density of 3.5 mA/cm 2 using the combination of chitosan/reduced graphene oxide/polyaniline as the electrode. Lv et al. [65] developed the carboxyl-functionalized mesoporous carbon electrode and used Nafion as an electron transfer medium. The carboxyl-functionalized mesoporous carbon electrode is optimized using oxidation time, concentration, and temperature as the main factors to increase enzyme activity and enzyme immobilization up to 140.72 and 242.74%, respectively. There are various electrodes with a high density of the hydroxyl and amino groups in chitosan, which have the advantage of biocompatibility suitable for use in EBFC, and Kim et al. [66] combined them with cobalt metal and graphene oxide as a composite electrode to immobilize the enzyme. The authors claimed that improved electrochemical performance in EBFC affects rheological and morphological properties due to the acidity pH of the chitosan.
Shakeel et al. [15] produced electrode material without binder or adhesive using a combination of reduced graphene oxide (rGO), functionalized magnetic nanoparticles (f-Fe 3 O 4 ), and polyaniline matrix. Biocompatible polymers with high conductivity, porosity, and surface area, together with the rGO and f-Fe 3 O 4 tested with appropriate enzyme loading, have increased electron transfer. The good electrical conductivity in graphene leads to the production of 3D graphene as a support for glucose oxidase with an extended enzyme lifetime and reduced leaching, developed by Babadi et al. [26]. The leaching is prevented by entrapping the enzyme at the 3D graphene surface and thus produces power at 164 µW/cm 2 . Three-dimensional graphene-related studies were also conducted by Werchmeister et al. [59] together with polyethylenimine (PEI) and ferrocene carboxylic acid (FcCOOH) using the drop-casting method. This study also tested the effect of Nafion solution on 3D graphene/PEI/FCCOOH on the carbon paper surface. The catalytic activity reduced by about 20% in the stability test, and the electrode can be stored for a week at a temperature of 4 • C. Butsyk et al. [67] synthesized the doped nitrogen and sulfur on carbon nano-onion as an enzyme support, in which the carbon nano-onion has properties of the graphene layer. The nitrogen and doped sulfur change the carbon-nano-onion capacitance characteristics and are suitable for the H 2 O 2 sensor in EBFC.
Reduced graphene oxide was dispersed on the 3D carbon paper electrode as a biocathode to minimize the aggregation problem of π-π stacking in reduced graphene oxide (rGO) by Tang et al. [68]. This r-GO/3D carbon paper combination provides enzyme orientation and confinement control to increase direct electron transfer efficiency with modification at the rGO surface through linker molecule 4-aminobenzoic acid (4-ABA). The bilirubin oxidase enzyme used in this study increased the half-life by 5 hours. Kuroishi et al. [69] used a modified electrode using the graphene-coated carbon fiber to help solve insufficient oxygen dissolved in the fuel liquid. Using graphene-coated carbon fiber with high porosity and surface area allowed the permeability of the oxygen gas to reach the electrode surface.
As stated earlier, the mediator is encouraged to be directly adsorbed on the electrode surface [28]. Shen et al. [70] took the initiative to produce the immobilized mediator redox protein cytochrome c (Cyt c) and used it on both anode and cathode. In addition, this material creates supercapacitor features that are for charge storage. The results show that the performance of EBFC was maintained at 80% when used as a 50 charge/discharge pulse. Kizling et al. [71] also developed an enzymatic biosupercapacitor using gold nanoparticlebased paper and nanostructured polypyrrole/nanocellulose as electrode materials with specific characteristics capacitance of 1.8 F/cm 2 . The supercapacitance gives one order magnitude of the current and power densities compared in the steady-state mode. A study by Niiyama et al. [44] modified the poly binder (vinylidene difluoride) and polytetrafluoroethylene (PTFE) on the MgO-templated carbon at the anode and cathode of the EBFC, respectively. The enzyme used will be supported by MgO/porous carbon. PTFE will control the hydrophobicity of the cathode so that the mass transport of oxygen gas becomes easy.
Gold (Au) electrode is commonly used in the EBFC cathode for oxygen reduction reactions (ORR). Wang et al. [20] produced self-powered sensors using EBFC to detect atrazine. To achieve use of the EBFC as a sensor, the authors used aptamers (Apt) supported on the Au electrode on the cathode section. ATZ was present at the cathode chamber and then bound at the Apt/Au electrode. The Apt/Au electrode has high sensitivity because it can detect ATZ as low as 7.5 mM due to the Au electrode helping to increase the electron transferability. Although a Pt metal catalyst showed good catalysis in ORR, Ji et al. [72] developed an iron and cobalt co-doped ordered mesoporous porphyrinic carbon (FeCo-OMPC) biocathode for cheaper ORR and achieved long-term stability. ORR activity increased with cobalt metal in the biocathode with a limited power density of 21.3 µW/cm 2 compared to without cobalt metal (9.6 µW/cm 2 ). Mazar et al. [73] used the combination of gold nanoparticle-reduced graphene oxide and a poly neutral red polymer composite electrode to interact with both enzymes at the bioanode and biocathode. Combining the gold nanoparticle-reduced graphene oxide increases the stability, selectivity, and high chemical structure by interacting with the poly neutral red polymer that enhances the electron transfer and thus operates the EBFC at 0.2 V and power of 3.6 µW/cm 2 . Kwon et al. [74] focused on cathode studies to improve EBFC performance by using tris-(2-aminoethyl) amine (TREN) as a linker for the connection between Au nanoparticles and the surface of CNT fibers. TREN is used to reduce electron transfer resistance because TREN is a short molecule compared to other linkers/binders. These layer-by-layer Au nanoparticles produce power as high as 1.2 mW/cm 2 .
The arrangement of the enzyme on the electrode surface plays a vital role so that the utilization of the enzyme can be maximized and provide excellent contact between the enzyme and the electrode surface. The work of Kizling et al. [75] resulted in cascade enzyme arrangement at the anode of the EBFC. In this study, various enzymes were used, namely mutarotase, invertase, fructose dehydrogenase, and flavine adenine dinucleotide (FAD)dependent glucose dehydrogenase in addition to using single or mixed substrates such as glucose, fructose, and sucrose. The comparison of other studies in this study shows that the resulting bioanode can produce a current as high as 2230 µA/cm 2 and maintain power generation for eight days before falling by 38%. Enzyme arrangement using nanoparticles that have a high surface area to volume ratio for enzyme loading [16] leads to a reduction in the distance of electron transport [75] and thus enhances the performance and stability of the EBFC [33]. EBFC can be applied as self-powered sensors to detect environmental pollutants. Pollutants such as atrazine are very harmful to humans and the environment, even at low ppb levels. Therefore, application of the EBFC as a biosensor electrode should have the ability to detect and quantify different analytes, good anti-interference performance [3], and lead to more efficient energy generation [42]. The diagram in Figure 6 shows the working schematic of the self-powered sensors using EBFC. Applications using EBFC as a biosensor need to develop electrodes using specific materials that can detect target molecules. Yu et al. [76] used EBFC for the detection of pathogenic bacteria. As shown in Figure 6, the aptamer is added in the developed system, and attached at the electrode surface, which is used to catch bacterial pathogens. Conventionally, in the absence of pathogens, glucose will be oxidized and generate electricity. The presence of pathogens will be trapped in the aptamer and will prevent glucose from oxidizing.
working schematic of the self-powered sensors using EBFC. Applications using EBFC as a biosensor need to develop electrodes using specific materials that can detect target molecules. Yu et al. [76] used EBFC for the detection of pathogenic bacteria. As shown in Figure 6, the aptamer is added in the developed system, and attached at the electrode surface, which is used to catch bacterial pathogens. Conventionally, in the absence of pathogens, glucose will be oxidized and generate electricity. The presence of pathogens will be trapped in the aptamer and will prevent glucose from oxidizing. Figure 6. Schematic illustration of EBFCs-based self-powered biosensor with the visual self-checking function for Vp determination. A) GDH would oxidize glucose to generate electrons, which could reduce blue PB to white PW; B) when connected with the biocathode electrode, the PW electrode would be oxidized to the original PB state and C) the color conversion between PB and PW realized the self-checking function via visual detection [76]. Copyright 2020 Elsevier.
Immobilization of laccase enzyme (LAC) using the encapsulation technique through the highly porous and large surface area of the metal-organic frameworks (zeolitic imidazolate framework-8, ZIP-8) for both the anode and cathode was carried out by Li et al. [3]. To increase biocompatibility in the EBFC electrode, LAC-ZIP-8 is supported on a combination of bacterial cellulose and CNTs. The resulting EBFC system can detect bisphenol A (BPA) linearly by increasing the concentration of BPA due to the good anti-interference performance improvement of the enzyme's thermal, organic solvent, and pH stability using the ZIP-8. Adachi et al. [60] introduced the platinum nanoparticle (Pt) in the enzyme-Au electrodes interaction to achieve the DET process. The enzyme used commonly requires a mediator, flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH). The Pt nanoparticle synthesized is located close to the enzyme, but the current difference produced without Pt is 1 mA/cm 2 . Wang et al. [12] introduced the SnS2 nanoflower/Au nanoparticles as a support material for thrombin detection. The presence of SnS2 nanoflower/Au nanoparticles has shown that the resulting electrode has capacitor Immobilization of laccase enzyme (LAC) using the encapsulation technique through the highly porous and large surface area of the metal-organic frameworks (zeolitic imidazolate framework-8, ZIP-8) for both the anode and cathode was carried out by Li et al. [3]. To increase biocompatibility in the EBFC electrode, LAC-ZIP-8 is supported on a combination of bacterial cellulose and CNTs. The resulting EBFC system can detect bisphenol A (BPA) linearly by increasing the concentration of BPA due to the good anti-interference performance improvement of the enzyme's thermal, organic solvent, and pH stability using the ZIP-8. Adachi et al. [60] introduced the platinum nanoparticle (Pt) in the enzyme-Au electrodes interaction to achieve the DET process. The enzyme used commonly requires a mediator, flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH). The Pt nanoparticle synthesized is located close to the enzyme, but the current difference produced without Pt is 1 mA/cm 2 . Wang et al. [12] introduced the SnS 2 nanoflower/Au nanoparticles as a support material for thrombin detection. The presence of SnS 2 nanoflower/Au nanoparticles has shown that the resulting electrode has capacitor characteristics and produces very high sensitivity detection (18.4 times) compared to a standard electrode. All developed electrode materials are summarized in Table 1.

System in EBFC
Various EBFC systems have been designed for the suitability of EBFC applications. Making the EBFC system is essential for the application involved. For example, to detect blood glucose, the EBFC system developed should be easy to use and dispose of [77]. Most EBFC systems exhibit decreased power and limited lifetime because solvent evaporation in the hydrostatic electrolyte and biofuel depletion require paper exchange replacement and electrolyte refilling [77]. To reduce external components such as pumps, Duong et al. [17] have produced self-pumping EBFCs, as shown in Figure 7. Most fuel cell systems try to minimize the use of external components because it increases the weight of the fuel cell. In addition, the EBFC system that uses a self-pumping technique is suitable for miniaturization applications, where it is ideal for use in portable, wearable, and implantable devices [23]. To produce the self-pumping EBFC system, the flow field produced is driven by the capillary effect, as shown in Figure 7, to supply the fuel, increase efficient mass transfer [75], and reduce fabrication cost and volume of microfluidic biofuel cell [78]. Duong et al. [63] further investigate the factor of electrode microstructure, electrochemical property, and enzyme loading using a self-pumping EBFC that can retain 89.2% on the stability test for 240 h. Rewatkar et al. [51] also developed a self-pumping EBFC but using the Y-shaped flow structured cell. Meanwhile, Mazar et al. [73] created a Y-shaped flow structured cell using the printed circuit board (PCB)/double-sided pressure-sensitive adhesive (PSA) method to fabricate the cell. The authors also stated that this method is more accessible than using the thermal-or plasma-coating method and photolithography. This Y-shaped microchannel provides an advantage in reducing the depletion boundary layer and cross-diffusional mixing between fuel and oxidant [79].

System in EBFC
Various EBFC systems have been designed for the suitability of EBFC applications. Making the EBFC system is essential for the application involved. For example, to detect blood glucose, the EBFC system developed should be easy to use and dispose of [77]. Most EBFC systems exhibit decreased power and limited lifetime because solvent evaporation in the hydrostatic electrolyte and biofuel depletion require paper exchange replacement and electrolyte refilling [77]. To reduce external components such as pumps, Duong et al. [17] have produced self-pumping EBFCs, as shown in Figure 7. Most fuel cell systems try to minimize the use of external components because it increases the weight of the fuel cell. In addition, the EBFC system that uses a self-pumping technique is suitable for miniaturization applications, where it is ideal for use in portable, wearable, and implantable devices [23]. To produce the self-pumping EBFC system, the flow field produced is driven by the capillary effect, as shown in Figure 7, to supply the fuel, increase efficient mass transfer [75], and reduce fabrication cost and volume of microfluidic biofuel cell [78]. Duong et al. [63] further investigate the factor of electrode microstructure, electrochemical property, and enzyme loading using a self-pumping EBFC that can retain 89.2% on the stability test for 240 h. Rewatkar et al. [51] also developed a self-pumping EBFC but using the Y-shaped flow structured cell. Meanwhile, Mazar et al. [73] created a Y-shaped flow structured cell using the printed circuit board (PCB)/double-sided pressure-sensitive adhesive (PSA) method to fabricate the cell. The authors also stated that this method is more accessible than using the thermal-or plasma-coating method and photolithography. This Y-shaped microchannel provides an advantage in reducing the depletion boundary layer and cross-diffusional mixing between fuel and oxidant [79]. Several researchers have developed microfluidic EBFCs [73,[80][81][82] without using the proton exchange membrane (PEM). Although this system successfully eliminates the use of expensive PEM, problems such as poor mass transport, mixing between fuel and oxidant, high internal resistance, insufficient fuel utilization, and interactive interference still occur [83]. Poor mass transport occurs when reactant/glucose slowly reaches the enzyme for the catalytic to happen. The system of microfluidic EBFC with cross-diffusional mixing was developed by Khan et al. [79]. This study examined the differences in flow rates and microchannel heights that lead to the optimal output of power density and current density at 153 µW/cm 2 and 450 µA/cm 2 at a flow rate of 25 mL/h and microchannel height of 450 µm, respectively. Gai et al. [83] introduced the nanocarrier in a membrane-less EBFC metal-organic framework [[Fe(CN) 6 ] 3 ] 3− to reduce the internal resistance in EBFC and enhance the power output 700 times. The ability of the enzyme to have high stability and selectivity is essential in EBFC. Therefore, the need to analyze the product resulting in the reaction at the EBFC electrode is needed. Varničić et al. [84] produced membrane-less EBFCs and used nuclear magnetic resonance spectroscopy to determine the production of two main products: D-arabinose and formic acid. The authors found that the production of this product was influenced by the enzyme selection on the cathode side of EBFC.
Needle-type EBFCs have also been developed to detect glucose by poking the needle on the skin. Two types of microneedles that have been used are polymeric needles and metallic hollow needles. Yin et al. [85] used needle-type EBFC to detect the glucose in apple, grape, and kiwifruit and gave the EBFC a value of 33, 55, and 44 µW, respectively. The resulting system had anti-biofouling 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and waterproof tape at the cathodic chamber to maintain power output and lifetime EBFC.
To expedite and facilitate EBFC cell fabrication with high precision and uniformity [40], 3D printing was used by Rewatkar et al. [81] due to the quick fabrication process, costeffectiveness, and simplicity. In this study, polylactic acid and conductive composite graphene materials were used to form microchannels and electrodes. The power and current of the EBFC produced using this fabrication are 4.15 µW/cm 2 and 13.36 µA/cm 2 at different flow rates, respectively. In other work by the same research group [80], they used non-toxic pencil graphite electrodes (PGE) as electrode materials in the 3D printing fabrication process. MWCNT coats the PGE as support for enzyme immobilization. The microchannel used in this study was a Y-shaped type that produced 18 µW/cm 2 at 0.433 V. The fabrication methods used in making EBFC are fused deposition modeling (FDM), paper-based, photolithography, laser micromachining, soft lithography, and xurography. Although various fabrication techniques can be chosen, advantages such as simple processing procedure, cost-efficiency, speedy production, high security, and durability are given priority [40].
Stacking fuel cells combine several cells to increase power. Yoshida et al. [86] combined six EBFC cells in series, as shown in Figure 8, and used fructose as fuel. This, combined with a very thin electrode suitable for the skin patch, increased the voltage and current by 1.5 V and 80 µA, respectively. Shitanda et al. [87] also combined six cells, but shapes such as hexagons were fabricated using screen printing. These cells were used to detect glucose in the urine, and the detectable glucose concentration was in the range of 1 to 25 mM. It seems that the fabrication of these two stacks shows that EBFC is easy to dispose of after use. Markovic et al. [88] also used the screen printing technique to produce a single cell of EBFC. Rewatkar et al. [78] developed the four series-parallel arrangements (series, parallel, and combined series-parallel) of microfluidic paper-based analytical EBFCs using a mini 3D-printed platform. In this study, the 4-series arrangement produced a stable open-circuit voltage of 1.65 V with a power density of 46.4 µW/cm 2 at 0.8 V. The feedforward control of the DC-DC PWM (pulse width modulation) boost converter was used in the power management circuit for stacking EBFC so that the power generated had a high power conversion efficiency. Seok et al. [77] studied the effect of EBFC power when changing the number and size of cells to optimize the DC output voltage. The efficiency of the DC-DC converter of 50% produced a capacity of 13 µW with an output voltage of 0.88 V on five stacks of EBFC. feedforward control of the DC-DC PWM (pulse width modulation) boost converter was used in the power management circuit for stacking EBFC so that the power generated had a high power conversion efficiency. Seok et al. [77] studied the effect of EBFC power when changing the number and size of cells to optimize the DC output voltage. The efficiency of the DC-DC converter of 50% produced a capacity of 13 µW with an output voltage of 0.88 V on five stacks of EBFC. Figure 8. The series connection of EBFC [86]. Copyright 2020 ACS Publication.

Conclusions
EBFC has advantages over other fuel cells, such as non-toxic chemical fuel, simplicity, low cost, small size, easy disposal, rapid start-up, eco-friendliness, anti-interference performance, and being easy to operate at room temperature and at neutral pH. However, the electrode materials used must have characteristics such as biocatalytic properties, biocompatibility, biodegradability, implantability, and mechanical flexibility in EBFC. Enzymes that become the heart of the EBFC need to immobilize on the surface electrode. Various methods such as polymer conduction, crosslinking, layer-by-layer assembly, and covalent attachment are used. The current trend mainly uses polymer conduction and crosslinking methods to immobilize the enzyme where the interaction between this material and the enzyme needs to be seen in terms of storage, solvent, pH, and thermal stability, function well, and have high efficiency. However, there is still inadequate and ineffective interaction between the materials used for enzyme immobilization, including imperfect and low enzyme loading, leaching out of enzyme that leads to degradation in longterm operation, potential enzyme poisoning when using non-biocompatibility support materials, reduced lifetime, and limited enzyme stability. Recent studies also use nanoparticle materials to increase electron transfer, although mediators are still used and have higher power performance in EBFCs. However, the mediator also has problems, such as the mediator itself being toxic in the EBFC environment, leakage, producing a low opencircuit voltage (OCV), mediator mobilization, high cost, and instability of the metal ionbased redox. Meanwhile, EBFC systems have various types depending on the use or application of EBFC, such as developing a self-pumping technique suitable for miniaturization applications, while the needle-type EBFCs have been designed to detect glucose poking the needle on the skin. The development of EBFC requires 3D printers due to the quick fabrication process, cost-effectiveness, and simplicity. Therefore, overall, among the combinations of materials that can be studied, namely nanoparticles, graphene, and conductive polymers, these three have the advantage of chemical stability and enhanced electron transfer, and the interaction between these materials can improve EBFC performance. The

Conclusions
EBFC has advantages over other fuel cells, such as non-toxic chemical fuel, simplicity, low cost, small size, easy disposal, rapid start-up, eco-friendliness, anti-interference performance, and being easy to operate at room temperature and at neutral pH. However, the electrode materials used must have characteristics such as biocatalytic properties, biocompatibility, biodegradability, implantability, and mechanical flexibility in EBFC. Enzymes that become the heart of the EBFC need to immobilize on the surface electrode. Various methods such as polymer conduction, crosslinking, layer-by-layer assembly, and covalent attachment are used. The current trend mainly uses polymer conduction and crosslinking methods to immobilize the enzyme where the interaction between this material and the enzyme needs to be seen in terms of storage, solvent, pH, and thermal stability, function well, and have high efficiency. However, there is still inadequate and ineffective interaction between the materials used for enzyme immobilization, including imperfect and low enzyme loading, leaching out of enzyme that leads to degradation in long-term operation, potential enzyme poisoning when using non-biocompatibility support materials, reduced lifetime, and limited enzyme stability. Recent studies also use nanoparticle materials to increase electron transfer, although mediators are still used and have higher power performance in EBFCs. However, the mediator also has problems, such as the mediator itself being toxic in the EBFC environment, leakage, producing a low open-circuit voltage (OCV), mediator mobilization, high cost, and instability of the metal ion-based redox. Meanwhile, EBFC systems have various types depending on the use or application of EBFC, such as developing a self-pumping technique suitable for miniaturization applications, while the needle-type EBFCs have been designed to detect glucose poking the needle on the skin. The development of EBFC requires 3D printers due to the quick fabrication process, cost-effectiveness, and simplicity. Therefore, overall, among the combinations of materials that can be studied, namely nanoparticles, graphene, and conductive polymers, these three have the advantage of chemical stability and enhanced electron transfer, and the interaction between these materials can improve EBFC performance. The development of chitosan selection in EBFC is also prevalent in publications because chitosan has the advantages of sensitivity, selectivity, stability, and reproducibility. Data Availability Statement: Data sharing is not applicable to this article as no new data were created or analyzed in this study.