Magnetic Nanoparticle-Reduced Graphene Oxide Nanocomposite as a Novel Bioelectrode for Mediatorless-Membraneless Glucose Enzymatic Biofuel Cells

In this work, an enzymatic biofuel cell (EBC) based on a membraneless and mediatorless glucose enzymatic fuel cell system was constructed for operation in physiological conditions (pH 7.0 and temperature 37 °C). The new platform EBC made of nanocomposite, including magnetic nanoparticles (Fe3O4 NPs) and reduced graphene oxide (RGO), was used for the immobilization of glucose oxidase (GOD) as bioanode and bilirubin oxidase (BOD) as biocathode. The EBC bioelectrodes were fabricated without binder or adhesive agents for immobilized enzyme and the first EBC using superparamagnetic properties with Fe3O4 NPs has been reported. The performance of the EBC was evaluated with promising results. In EBC tests, the maximum power density of the EBC was 73.7 μW cm−2 and an open circuit voltage (OCV) as +0.63 V with 5 mM of glucose concentration for the physiological condition of humans. The Fe3O4-RGO nanocomposite offers remarkable enhancement in large surface areas, is a favorable environment for enzyme immobilization, and facilitates electron transfer between enzymes and electrode surfaces. Fe3O4 and RGO have been implied as new promising composite nanomaterials for immobilizing enzymes and efficient platforms due to their superparamagnetism properties. Thus, glucose EBCs could potentially be used as self-powered biosensors or electric power sources for biomedical device applications.


Results and Discussion
Characterization of Fe 3 O 4 -RGO/GOD nanocomposite. The bioelectrodes fabrication was shown as schematic in Fig. 1. Due to Fe 3 O 4 NPs being surrounded by a positive charge, they play an important role in immobilizing enzymes through electrostatic interaction. GOD is a negatively charged biomolecule at pH 7.0 23 that can be easily immobilized onto the positively charged amino group on Fe 3 O 4 NPs surface via electrostatic interaction. Figure 2A, the resultant Fe 3 O 4 -RGO nanohybrids could be immediately separated from the mixture when a magnet was placed nearby the glass vial within 30 second resulting in a clear and transparent solution. Thus, the attraction and dispersion processes can be readily altered by applying and removing an external magnetic field. The morphology of Fe 3 O 4 -RGO and Fe 3 O 4 -RGO/GOD nanocomposite was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 2B shows a TEM image of Fe 3 O 4 -RGO. It can be seen that Fe 3 O 4 NPs was distributed on the RGO sheet revealing a stacked, crumpled, wrinkled and rippled structure. Particle size was estimated to be in the range of 15-32 nm. GOD was immobilized on the Fe 3 O 4 -RGO nanocomposite by electrostatic force, as shown by the SEM image in Fig. 2D, where Fe 3 O 4 -RGO/ GOD appears the spherical Fe 3 O 4 NPs were roughness and the particles size increased dramatically and is clearly observed compared with SEM image of Fe 3 O 4 -RGO in Fig. 2C. This indicated that the protein globular structure of GOD was uniform on the Fe 3 O 4 -RGO structure. Figure 3A shows the Raman spectrum of Fe 3 O 4 -RGO, which exhibited peaks at 1371 cm −1 and 1591 cm −1 that correspond to the D band of the breathing mode of k-point phonons of A1g symmetry and G-band of the first-order scattering of the E2g phonons 24 , respectively. The intensity ratios I D /I G of Fe 3 O 4 -GO and Fe 3 O 4 -RGO were found to be 1.06 and 1.13, respectively, indicating the greater sp 2 characteristic of graphene. This increase of I D /I G ratio is due to the decrease of the sp 2 in-plane domain induced by the introduction of defects and disorder of the sp 2 domain. This indicates that sp 2 domains of Fe 3 O 4 -GO are formed during reduction using glucose as the reducing agent. FT-IR spectra of Fe 3 O 4 -RGO, Fe 3 O 4 -RGO/GOD and GOD are presented in Fig. 3B at curve a, b and c, respectively. For the Fe 3 O 4 -RGO, no obvious absorption peak was observed. The FTIR spectrum of native GOD shows two characteristic peaks at 1654 and 1545 cm −1 , which are attributed to amide I and II bands of protein that can provide detailed information on the secondary structure of the polypeptide chain 25,26 . The band at 1104 cm −1 was C-O bond stretching. The broad and strong peak at 3299 cm −1 was assigned to hydroxyl (OH) stretching vibrations 27 Fig. S1. The Fe 3 O 4 -NH 2 contain large amount of amine groups at neutral pH in DI water. The amine functionalized Fe 3 O 4 nanoparticles showed a positive zeta potential of 30.6 mV due to the due to the protonation of its -NH 2 group on the surface. The Fe 3 O 4 -NH 2 was covalent chemical bond with the carboxylic group of the GO present as Fe 3 O 4 -GO. The zeta potential of −16 mV for Fe 3 O 4 -GO can be explained by the presence of bulky oxygen groups such as carboxyl groups on GO sheet. After reduction process of GO to RGO, the zeta potentials of Fe 3 O 4 -RGO was 18.5 mV due to positively charge of Fe 3 O 4 on RGO surface. The isoelectric point (pI) of GOD is 4.2, which reveals that GOD carries net negative charges at pH 7.0. The results showed that GOD is negatively charged at pH 7.0 which corresponding to zeta potential of GOD in PBS  Electrochemical behavior of magnetic glassy carbon electrode (MGCE). The electrochemical behavior of fabricated magnetic glassy carbon electrode (MGCE) was characterized by cyclic voltammetry. Ferricyanide (K 3 Fe(CN) 6 ) was used as a redox probe to investigate the electrochemical behaviors of MGCE comparing to the bare commercial glassy carbon electrode (GCE) with equal diameter of 3 mm at the scan rate of 50 mV/s in 0.1 M PBS pH 7.0. Figure 4A, the CVs curve of the bare GCE in curve a showed a pair of well-defined quasi-reversible peaks with slightly lower peak current and larger peak separation potential than MGCE in curve b. Compared to the bare GCE, the redox peak currents of the bare MGCE increased greatly, implying that the MGCE electrochemical property can be applied for biosensors and BFCs. The electrode surfaces of glassy carbon have been examined by microscopy. Figure S2a-c shows microscopic images of unpolished, polished bare MGCE and bare GCE surface, respectively. It can be seen that the unpolished bare MGCE was rough. After applied aluminium oxide particles to give a polished MGCE surface, the electrode surface was smooth without scratches surface.

Direct electrochemistry of GOD immobilized Fe 3 O 4 -RGO modified electrode. GOD molecules
have flavin adenine dinucleotide (FAD) as redox centers deep localization inside the protein structure, thus the DET for GOD is extremely difficult. In order to improve the electron transfer of FAD, Fe 3 O 4 -RGO was applied to immobilize GOD. Figure 4B   the standard electrode potential of −0.483 (vs. Ag/AgCl) for FAD/FADH 2 at pH 7.0 28 , suggesting that the GOD molecules retain bioactivity after adsorption on the Fe 3 O 4 -RGO nanocomposites. The DET process mechanism was described in equation (1) and (2). GOD is two protons and two electrons coupled reaction, FAD serves as the catalytic site of GOD by accepting the electrons donated by the glucose and being reduced to FADH 2 . In this process glucose is converting into gluconolactone. (GOD)FADH 2 is then oxidized by electrode to (GOD)FAD. Two protons and two electrons can subsequently be transferred from GOD to bioanode. O 2 is a natural electron acceptor for GOD. In presence of O 2 , GOD can be transfer electron to O 2 then reduced into hydrogen peroxide as present in equation (3). Therefore, O 2 is a competing electron acceptor to DET reaction. Unfortunately, it is well know that DET system did not require oxygen due to FAD serves as the catalytic site of GOD by accepting the electrons donated by the glucose and being reduced to FADH 2 . The electrons can be transferred directly from GOD to electrode through Fe 3 O 4 -RGO composite. Moreover, based on membraneless EBC, the cathode compartment could be consumed most of O 2 for reduction reaction. In addition, the stability of Fe 3 O 4 /GOD/MGCE was also evaluated as shown in Fig. S3. There was no obvious change in redox peaks could be seen from the CV curves, the CVs curves still almost remained from their initial cycle after continuous scanning for 100 scan cycles. This can be implied that the fabricated electrode is very stable.
The influence of scan rate. The effect of the scan rate on cyclic voltammetric performance at the Fig. 5A. The redox processes of nanocomposite gave almost symmetric anodic and cathodic peaks (E pa and E pc ) at relatively slow scan rates. When the scan rate increases, the redox potentials of GOD shift slightly. The anodic and cathodic peak currents linearly increased with the increasing scan rate from 10 to 100 mV/s. This indicates that the redox reaction of GOD on Fe 3 O 4 -RGO modified electrode was a quasi-reversible surface-controlled process. The surface concentration (Γ, mol/cm 2 ) of electroactive GOD can be calculated as 2.03 × 10 −11 mol/cm 2 according to the formula Γ = Q/nFA, where Q is the charge consumed in C, n is the number of electrons transferred (n = 2), A is the electrode area (cm 2 ) and F is the faraday constant. Figure     Electrocatalytic behavior of biocathodes. For biocathodes, the reduction of O 2 generally utilized two types of enzymes, including bilirubin oxidase and laccase. Laccase presents an optimum activity around pH 4-5 30 . BOD electrocatalytic activity was investigated at neutral pH or pH 7.0 which was suitable for a real application system. BOD is one of the multicopper oxidase selected for catalyzing the four-electron reduction of oxygen to water at the biocathode because BOD can efficiently work as an electrode biocatalyst even under neutral conditions. In their structure, the T1 copper site gives electrons to the electrode and transfers those electrons to the T2/ T3 copper site 31 , where oxygen is reduced to water in a four-electron transfer mechanism according to Equation 5. The electrocatalytic reaction of BOD on Fe 3 O 4 -RGO/MGCE as biocathode was examined using CV. The experiments were carried out under nitrogen saturated and oxygen saturated conditions at the potential between −0.1 V to + 0.7 V at a scan rate of 1 mV/s. Figure 7A displays the CVs recorded at Fe 3 O 4 -RGO/BOD/MGCE in N 2 (curve a) and O 2 (curve c) saturated 0.1 PBS pH 7.0. It can be seen that the presence of oxygen in the system and a highly enhanced cathodic peak current increase was observed with a peak potential of oxygen reduction of +0.51 V versus Ag/AgCl. The potential of the peak begins at +0.60 V, whereas the presence of nitrogen in the modified electrodes exhibited no catalytic activity. These results agree with literature data of approximately +0.5 V (vs. Ag/AgCl) 32 , which is close to the redox potential of the T1 copper site BOD. It demonstrates that the modified electrode that immobilized BOD has the capability to achieve DET and efficiently catalyze oxygen reduction to water. Linear sweep voltammetry (LSV) was also used to study the electrocatalytics of RGO-Fe 3 O 4 /BOD/MGCE. Figure 6B shows   Table 1. The EBC showed repeatability with an R.S.D of 5.73% for 5 repeatable measurements carried out with the flow system. These results indicate that Fe 3 O 4 -RGO based nanocomposites can be useful materials for the fabrication of EBC to gain energy from biological fuels such as glucose. Moreover, Fe 3 O 4 -RGO has great potential for the fabrication of glucose EBC due to operation in the  physiological conditions of humans, preparation protocols and simple EBC assembly protocols without mediators or membranes.
The stability of EBC. The stability of EBC was characterized by measuring its power loss when continuously working in an air-saturated quiescent buffer containing 5 mM glucose coupling with 1 MΩ of resistance loaded on the cell. The maximum power density was observed for 4 weeks. After operating for 24 h, the power of the EBC retained 98.37 % of its original power output and held steady at 95.39 % after duration of 7 days. Then 78.7 % of initial power density was retained even after 4 weeks, which revealed good durability and stability of the fabricated EBC as shown in Fig. S4. However, the OCV of the EBC remained unchanged during the duration. This could indicate that covalent bonding was unaffected by changes in the surrounding environment. Moreover, the electrostatic interaction binds with enzymes and magnetic force at MGCE to prevent leaching from the electrode surface during operation and storage. In EBC development, the stability of the enzymes on the electrode is the main factor for retaining long-term performance of the membraneless EBC. However, no significant breakthrough has been achieved in this work concerning the longevity of EBCs. Magnetic glassy carbon electrode fabrication. The magnetic glassy carbon electrode (MGCE) was prepared by placing a nummular NdFeB magnet (3 mm in diameter and 4 mm in thickness) on a glassy carbon (3 mm in diameter and 3 mm in thickness). A copper wire was put around the magnet and filled with silver glue, which was then put into an acrylic tube (10 mm in diameter and 20 mm in depth). All components were fixed with epoxy resin then cured at room temperature for at least 24 h. The MGCE was successively polished with emery paper and alumina powder, followed by sonication in DI water.

Conclusions
This paper successfully demonstrates a design and simple platform for construction of an enzymatic biofuel cell based on direct electron transfer (mediatorless) BFC by Fe 3 O 4 -RGO/GOD as the bioanode and Fe 3 O 4 -RGO/BOD as the biocathode. Enzymes were incorporated into Fe 3 O 4 -RGO by strong electrostatic interaction. The properties of graphene and magnetic nanoparticles enhance enzymatic biofuel cells for more efficient conductivity and also increase the immobilization of enzymes and modified bioelectrodes without the binder or adhesive agents that usually block electron transfers at electrode surfaces. Fe 3 O 4 NPs not only increases the surface area, but also has paramagnetic properties which make them more easily manipulated by an external magnetic field to prevent the leakage of enzymes at electrode surfaces. This bioelectrode fabrication approach could offer promising solutions for generations of new classes of membraneless biofuel cells.