Applications of chitosan (CHI)-reduced graphene oxide (rGO)-polyaniline (PAni) conducting composite electrode for energy generation in glucose biofuel cell

A glassy carbon electrode (GC) immobilized with chitosan (CHI)@reduced graphene (rGO)-polyaniline (PAni)/ferritin (Frt)/glucose oxidase (GOx) bioelectrode was prepared. The prepared electrode was characterized by using cyclic voltammetry (CV), linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) techniques. The morphological characterization was made by scanning electron microsopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. This bioelectrode provided a stable current response of 3.5 ± 0.02 mAcm−2 in 20 mM glucose. The coverage of enzyme on 0.07 cm2 area of electrode modified with CHI@rGO-PAni/Frt was calculated to be 3.80 × 10−8 mol cm−2.

www.nature.com/scientificreports www.nature.com/scientificreports/ glutaraldehyde (Glu) were purchased from Sigma-Aldrich, India. Phosphate buffer saline (PBS) pH 7.0 and 5.0, GOx having activity from 100,000 to 150,000 units g −1 protein supplied by Central Drug House, India. Glucose was purchased from Himedia and Chitosan with a deacylation degree >90% was received from SRL, India. Double distilled water (DDW) was utilized throughout the investigations. All the materials were of analytical grade and used as received without further purification.
instrumentation. The voltammetric experiments and electrochemical impedance spectroscopy (EIS) were performed to check the electrochemical performances of the developed electrodes by using a computer controlled Potentiostat/Galvanostat (PGSTAT 302N Autolab) under nitrogen purging. The cell assembly consists of a conventional three-electrode system using a platinum wire electrode as a counter electrode (CE), KCl saturated Ag/AgCl reference electrode (RE) and glassy carbon electrode (GC) modified with CHI@rGO-PAni/Frt/ GOx as a working electrode (WE) having a 0.07 cm 2 surface area. The electrochemical impedance spectroscopy (EIS) experiments were recorded in a 0.1 M potassium chloride solution comprising of 2.0 mM K 4 [Fe(CN) 6 ] in a frequency range from 0.1 to 10 kHz. The ultrasonicator was used for the partial cleaning of the electrode with ethanol. The morphological and chemical characterizations for the nanocomposite were carried out by FEI Quanta 250 FEG scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy recorded by Bruker (Platinum ATR-QL diamond system) in the spectral range from 4000 to 500 cm −1 . All the experiments were conducted at room temperature.
preparation of the composite material. rGO synthesis. The graphene oxide (GO) was synthesized by Hummer's method 70  Synthesis of rGo-pAni composite through in-situ polymerization. A 1.4 g of 5-sulfosalicylic acid (dopant) was dissolved in 200 mL water. A 0.5 g of rGO and 2 mL of aniline were added in it. The solution obtained was continuously agitated until a black homogeneous dispersion was formed which is further placed on an ice bath by maintaining the temperature below 6 °C. A solution of APS (5.02 g) was prepared in 100 mL of water by vigorous stirring. This solution was added dropwise into the mixture of rGO and aniline. After some time, the resulting solution turned into blackish-green in color, which is an indication of the formation of emeraldine salt of polyaniline. The precipitate obtained was carefully washed with EtOH and H 2 O constantly until a clear solution was obtained. The precipitate was dried at 50 °C 71,72 . cHi@rGo-pAni synthesis. A solution was prepared by dissolving 0.5 g of CHI in 50 mL of aqueous CH 3 COOH (2% v/v) by stirring it for 8 h. In that, rGO-PAni was dispersed by ultrasonication for 60 min. Figure 1 displays the mechanism of the composite formation in which the rGO sheets interact with the SSA doped PAni. The interaction of rGO and doped PAni occurred via hydrogen bond between phenolic OH and PAni radical (protonated by SSA). These interactions clutch the rGO sheets and PAni matrix together. However, the stronger bonds are the ones formed by electrostatic interactions originating from protonated N and lone pairs on OH group. The π-π stacking between the PAni and rGO rings further stabilizes the complex structure of the composite. Further, the rGO-PAni composite interacts with the protonated CHI via hydrogen bonding and electrostatic interaction that makes the huge structure of ternary composite stable.
preparation of the bioanode. The CHI@rGO-PAni/Frt/GOx bioelectrode was constructed as follows; first, the well-polished glassy carbon (GC) electrode was ultrasonicated for 10 min and rinsed with double distilled water. Further, the surface of the electrodes was cleaned by CV in 1.0 M sulfuric acid at a 50 mVs −1 potential sweep rate via the Autolab (PGSTAT 302N, Metrohm). The CHI@rGO-PAni dispersion was loaded on four GC electrodes with an amount of 4.0, 6.0, 8.0, and 10.0 μL. The CVs were taken for the sake of optimization. It was found that the electrode with 8.0 μL offered a better background current. The CHI@rGO-PAni modified three GC electrodes were altered with different amounts of Frt (2.0, 4.0, and 6.0 μL). It was observed from a CV curve that the electrode with 4.0 μL was performing better among the three modified electrodes. In the same manner, GOx solution (10.0 mg mL −1 GOx in 0.1 M sodium phosphate buffer saline of pH 5.0) was optimized for 4, 8, and 10 μL on the three GC electrodes with the pre-optimized materials. The CHI@rGO-PAni/Frt electrode loaded with 10 μL of GOx solution performed well than the other altered electrodes. Finally, the optimized GC/CHI@ rGO-PAni/Frt/GOx bioanode was dried at room temperature. A 2.0 μL of 2% glutaraldehyde aqueous solution was drop cast to stabilize and crosslinked the CHI@rGO-PAni nanomaterial with Frt and GOx on the electrode surface.

Results and Discussions
Scanning electron microscope (SeM) analysis. The scanning electron microscopic morphologies of rGO-PAni, CHI@rGO-PAni, and CHI@rGO-PAni/Frt/GOx composites are displayed in Fig. 2(a-d). A fibrillar matrix of short granular structures can be seen in Fig. 2a. However, in the case of CHI@rGO-PAni, a granular matrix having an uneven distribution of pores can be seen which confirmed the disappearance of fibrous features when rGO-PAni was ultrasonicated in the solution of CHI. Interestingly, a porous structure can be observed in Fig. 2b, which is quite advantageous for the loading of mediator Frt with the immobilization of GOx. However, Fig. 2 c and d showed the successful capping of pores with the Frt and GOx. Besides, the porous structure allows the movement of the fuel towards the surface of the electrode for the redox reaction to occur. www.nature.com/scientificreports www.nature.com/scientificreports/ fourier transform infrared (ftiR) analysis. The FTIR spectra of CHI, rGO-PAni and CHI@rGO-PAni composites are exhibited in Fig. 3. The important peaks of these composites are mentioned in Table 1. The intense broad peak at 3425 cm −1 is attributed to the overlying of O-H from SO 3 H with N-H characteristic stretching 35,61,62 . The significant peaks of PAni because of C = C ring stretching vibration of quinoid and benzoid in rGO-PAni and CHI@rGO-PAni could be found at 1487, 1491 cm −1 and 1571, 1584 cm −1 , respectively. The peaks at 2853 and 2920 cm −1 in rGO-PAni and peaks at 2852 and 2918 cm −1 in CHI@rGO-PAni are due to C-H stretching. All the characteristic peaks of PAni-rGO and CHI are visible in the FTIR spectrum of a ternary composite. Though, the little shift was observed which might be due to the hydrogen bond among the components of the ternary composite 73 . Furthermore, the peaks found at 1008, 10028 and 1008, 1032 cm −1 in the spectra of rGO-PAni and CHI@ rGO-PAni might be due to the presence of rGO in both of the composites. electrochemical activity of prepared electrodes.   www.nature.com/scientificreports www.nature.com/scientificreports/ with the CHI@rGO-PAni/GOx bioelectrode. This indicates that the efficiency of electron movement from deeply buried redox cofactor (∼13 Å) within the GOx to the conducting support CHI@rGO-PAni was enhanced by the mediator Frt. As the previous chemical and electrochemical researches on Frt have shown that the proteinaceous casing may behave as an electron conductor and mineralized core enhances the electronic conductivity of protein 26,27 . A pair of quasi-reversible redox peaks were observed on curves (b) and (c). These results indicated that the CHI@rGO-PAni/Frt/GOx ternary bio-nanocomposite might have the electrocatalytic activity towards the reduction of GOx cofactor FAD to FADH 2 74 . Figure 5 reveals the bioelectrocatalytic activity of the CHI@rGO-PAni/Frt/GOx in 0.1 M PBS with (20 mM glucose) and without glucose in the potential window of −1.7 to −0.7. The CV of the CHI@rGO-PAni/Frt/ GOx bio-nanocomposite exhibited a significant catalytic redox function with the generation of anodic current i.e. 2.5 ± 0.04 mA cm −2 , which is relatively more than the current produced by the CHI@rGO-PAni composite material. However, the adding of glucose in PBS triggered the much magnification in the current response up to 3.5 ± 0.02 mA cm −2 accompanied by the appearance of obvious quasi-reversible oxidoreduction peaks in the CV of the CHI@rGO-PAni/Frt/GOx. The pair of redox peaks appeared due to the conversion of the cofactor of GOx, i.e., FAD/FADH 2 , while the conversion of glucose to gluconolactone. This suggests that the redox reaction occurring at the modified electrode surface was intervened with the redox mediator Frt, and the required conducting platform was offered by the conducting CHI@rGO-PAni nanocomposite. Despite, with the help of Frt, the conducting properties and the suitable capacitive behavior of the nanocomposite concurrently brought the efficient electron transfer from the profoundly seated redox-active cofactor of GOx at the interface of electrode and electrolyte 33,46,75 .
The clear redox peaks with an apparent formal peak potential (average of anodic and cathodic peak potentials) at −0.45 V was calculated, which is near to the standard electrode potential of FAD/FADH 2 (−0.50 V) at pH 7.0 76 . This indicates the bioactivity of the GOx retained after immobilization on the CHI@rGO-PAni/Frt. The peak to peak separation (ΔE = E pa -E pc ) at 0.428 V vs. Ag/AgCl reference electrode was observed at 100 mVs −1 for CHI@rGO-PAni/Frt/GOx in the presence of glucose. This indicated the fast-mediated electron transfer of GOx, owing to the uniform porous structure and large specific surface area of the CHI@rGO-PAni/Frt biocomposite. These findings demonstrate that the as-prepared nanocomposite assists the beneficial immobilization of the GOx because of the high surface area, excellent electrocatalytic activity, and the suitable microenvironment provided  www.nature.com/scientificreports www.nature.com/scientificreports/ by the rGO-PAni grafted on CHI. Hence, it is confirmed that the fabricated bioanode is a potential candidate to be employed in the construction of glucose-based EFCs.
The influence of various potential sweep rates on the cyclic voltammetric performance of CHI@rGO-PAni/ Frt/GOx bioanode was recorded. Figure 6 shows that the response of peak currents gradually amplified with the increasing sweep rate along with the shifting of oxidation and reduction peaks in the right (positive) and left (negative) direction, respectively. Which assured the linear dependence of oxidation peak current (Ipa) and reduction peak current (Ipc) with linear regression equations I pa = 0.00045 v -0.0001, R 2 = 0.99 and I pc = −0.00032v-0.0008, R 2 = 0.98 at the scan rates (10-100 mVs −1 ), hence implying a quasi-reversible and surface-controlled electrochemical process 77,78 . The CVs in a broad spectrum of scan rates evident the significant electrochemical behavior of the developed bioanode.
The relation of redox peak potentials with the Napierian logarithm of the scan rates was analyzed to evaluate electrochemical parameters by utilizing the equations given below 79 :  www.nature.com/scientificreports www.nature.com/scientificreports/ Where E f is the formal potential, α is the charge transfer coefficient of the system, v is the scan rate, n is the number of electron transfer, k s is the heterogeneous electron transfer rate constant, T, R, and F have their usual meanings.
The n and α were calculated to be 1.88 and 0.50, respectively, from the slope of the lines of Fig. 7. The Laviron equation was applied to calculate the rate constant of heterogeneous electron transfer (k s = 0.50 ± 0.01 s −1 ) of the CHI@rGO-PAni/Frt/GOx modified electrode 80 . The k s of CHI@rGO-PAni/Frt/GOx was slightly lower than the similar work has reported which is due to the larger peak potential separation.
The surface coverage concentration of the bioelectroactive GOx, I * is estimated from the integration of the charge of the anodic peak in the CV curve in accordance with the Faraday law, Q = nFAI * , where Q is the charge integrated from the cathodic peak, A is the effective surface area of the electrode in cm 2 , F is the Faraday constant, and I * is the surface coverage of the enzyme GOx. I * of GOx on CHI@rGO-PAni/Frt. It was calculated to be 3.80 × 10 −8 mol cm −2 , which is comparatively four times higher than the bare GC electrode (2.86 × 10 −12 mol cm −2 ) 81 . This indicates saturated adsorption of GOx in CHI@rGO-PAni/Frt bionanocomposite. electrochemical impedance spectroscopy (eiS). EIS is a powerful technique for the investigation of impedance changes occuring during the redox reaction. The modified surface of the electrodes was characterized by EIS to analyze the electronic transfer properties of the composite materials at the interface of electrode and electrolyte 82 . This technique was used to confirm the interaction among rGO-PAni, CHI@rGO-PAni and CHI@ rGO-PAni/Frt/GOx tailored electrodes. The Nyquist plots displayed a semi-circle region with different diameters and a linear region, as shown in Fig. 8. The semi-circle region observed at larger frequencies indicates the electron transfer limited process and the linear region at smaller frequencies designates the diffusion process. The resistance to electron transfer (Ret) at the surface of the electrode can be computed through the semi-circle diameter. The diameter of the semi-circle region decreased among the samples, with CHI@rGO-PAni/Frt/GOx > CHI@  Linear sweep voltammetry (LSV) study. The LSV investigation was conducted to study the bioelectrocatalytic oxidation response generated by the CHI@rGO-PAni/Frt/GOx modified bioanode relating to the various concentration of glucose in PBS of pH 7.0 ( Fig. 9(A)). The electrocatalytic oxidation of glucose began at −0.46 V (E onset). The current density generated at anode raises with the addition in the glucose concentration upto 30 mM beyond which the current density got saturated because of the hindrance caused by the higher glucose concentration 29,30 . Hence, the catalytic current remains constant over time with the addition of more amount of glucose.
The current response obtained from the modified anode versus the various glucose concentrations is plotted in Fig. 9(B). The current density amplified from 2.3 ± 0.02 mA cm −2 for 5.0 mM glucose to 2.9 ± 0.02 mA cm −2 for 10 mM glucose and this then reached to the equilibrium at 3.5 ± 0.02 mA cm −2 for 20 mM glucose at 100 mVs −1 . This demonstrates the appropriate biocatalytic oxidation of glucose on the GC/CHI@rGO-PAni/Frt/GOx electrode. The saturation in current density accomplished by the ternary nanocomposite modified electrode is comparable with the other previously tested anodes as shown in Table 2. Hence, this research broadens the outlook of the real-world application to develop enzyme-catalyzed high-performance EFCs.
Stability. The stability of CHI@rGO-PAni/Frt/GOx and rGO-PAni/Frt/GOx bioanodes was also studied via electrochemical investigation. These two electrodes were stored at 4 °C and CVs were recorded periodically at 1 nd , 4 th and 7 th day. It was observed that initially, the rGO-PAni/Frt/GOx bioanode provided higher current than CHI@rGO-PAni/Frt/GOx, but with time the current density falls rapidly for the same electrode, as shown in Fig. 10. However, CHI@rGO-PAni/Frt/GOx bioanode showed lesser current density but better storage stability after one week and it retained 95% of its initial current response. These results exhibited that the mediated electrochemistry of GOx loaded on the CHI@rGO-PAni/Frt has good stability owing to the properties endowed by the integration of rGO-PAni on CHI which provided mechanical durability to the nanocomposite. Besides, the high surface area of the nanocomposite offered a large number of active sites to hold more biocatalysts. Therefore, it is clear that CHI@rGO-PAni/Frt/GOx bioanode has significant electrochemical stability.   www.nature.com/scientificreports www.nature.com/scientificreports/ conclusion The synthesized CHI@rGO-PAni biocomposite was used to construct a bioanode for glucose-based EFCs application showed good electrochemical properties along with substantial stability due to the collaborative effects among CHI, PAni, and rGO. The mediator Frt possessing the redox activity smoothed the movement of the electrons between the profoundly seated GOx active sites and the electrode surface. To ensure the mediated electrocatalytic activity, CV was performed and the resulting voltammograms suggest that the immobilized enzyme showed good bioelectrocatalytic activity for glucose oxidation. The prepared bioanode proved capable of generating a maximum current (3.5 ± 0.02 mAcm −2 ) for the oxidation of 20 mM glucose. The improvement of the performance was due to the porosity and large surface area of the anode material permitting higher loading of active enzymes and ease of fuel diffusion through the CHI@rGO-PAni customized electrode. The CHI@rGO-PAni/ Frt/GOx bioanode showed lesser current density but better storage stability after one week and it retained 95% of its initial response. The involvement of CHI enriches the biocompatibility of the prepared electrode which is beneficial for implantable EFCs. This study explained the potential of the prepared anode for the possible development of improved EFC performance. Future work will focus on the maximum utilization of the CHI along with conducting material to enhance long-term stability accompanying high electron transfer efficiency.