# Research Progresses and Application of Biofuel Cells Based on Immobilized Enzymes
## 2 A Variety of Carrier Materials for Enzyme Immobilization in EBFCs  
### Some Nanomaterials and Nanocomposites for Enzyme Immobilization in EBFCs  
EBFCs require enzyme immobilization materials with high surface area, high enzyme loading density, high adsorption durability and recyclability. In the technology of EBFCs, the enzyme immobilization material, that is, the carrier material for enzyme immobilization, is also an electrode material or electrode modification material; thus, its conductivity is also a key factor [1; 2]. Enzyme immobilization materials include those carbon-based nanomaterials with good electrical conductivity, large specific surface area and good biocompatibility, allowing large enzyme loading densities and comfortable microenvironments for the enzymatic reaction. Some flexible carbon materials such as buckypaper (BP), carbon paper (CP), graphene paper (GP), carbon cloth (CC), due to their good mechanical maneuverability and portability, good electrical conductivity, and good biocompatibility, are also widely used in EBFCs. Moreover, some conductive polymers such as polyaniline (PANI), polypyrrole (Ppy), polyindole (PIn), and various metal nanoparticles and inorganic nanoparticles can also improve the efficiency of EBFCs. Carbon nanotubes (CNTs), graphene oxidase (GO), and other carbon-based nanomaterials are generally hydrophobic, while enzymes require a hydrophilic microenvironment to remain active. Functionalization and modification using water-soluble polymers, pro  
Figure 1: Principles and configurations of enzymatic biofuel cells. teins, and hydrophilic inorganic materials can improve the hydrophilicity of carbon-based nanomaterials. [PERSON] et al. functionalized single-walled carbon nanotubes (SWCNTs) using H\({}_{2}\)N-DNA to obtain DNA@SWCNTs/GOx anodes and cathodes of SWCNTs/Lac, forming single-chambered diaphragmless EBFC [11]. The EBFC had a stable output of 730-760 \(\upmu\)W cm\({}^{-2}\) over a week [11]. Ferritin (Frt), a widely available iron storage protein, comprises a hydrated iron oxide core with a nanometer size and a protein shell with a cage-like structure. [PERSON] and [PERSON] et al. functionalized SWCNTs with Frt to obtain SWCNTs/Frt/GOx bioanodes [12]. The resulting EBFC achieved a current density of 4.6 mA cm\({}^{-2}\) in 20 mM glucose solution. Several other research groups also used Frt as a biocompatible material for the electrode construction of EBFCs [13; 14; 15; 16; 17]. [PERSON] et al. developed a bioenzymatic electrode using GOx and Catalase (Cat) as dual anodic enzymes and Lac as a cathodic enzyme, which were mechanically compressed with multi-walled carbon nanotubes (MWCNTs) to make a disc electrode [18]. The inclusion of Cat can eliminate the anodic by-product hydrogen peroxide and can resupply the anodic region with oxygen. The resulting bioenzymatic electrode of the cell showed high current densities for both glucose oxidation at the anode and oxygen reduction at the cathode, with an open-circuit voltage of 0.95 V and a maximum power density of 1.3 mW cm\({}^{-2}\). Under simulated physiological conditions (with 5 mM of glucose), the cell can operate stably for 1 month at 1 mW cm\({}^{-2}\). This simple method of compressing MWCNTs can be fully extended to other types of enzymes, which can guide the construction of electrodes for other types of biofuel cells subsequently [18]. Based on this method of making electrodes by mechanical compression, [PERSON] et al. also designed a cell that utilizes both indirect and direct electron transfer, using naphthoquinone (NQ)-mediated GOx oxidation of glucose at the anode for indirect electron transfer and Lac-catalyzed direct electron transfer at the cathode for direct reduction of oxygen [19]. The battery had an open-circuit voltage of 0.76 V, a current density of 4.47 mA cm\({}^{-2}\), and a maximum power density of 1.54 mW cm\({}^{-2}\). At 0.5 V discharge, the constant output power of the cell was 0.56 mW h cm\({}^{-2}\).