Optimization of rgo-pei/naph-sh/agnws/frt/gox nanocomposite anode for biofuel cell applications

ABSTRACT The present study reports a new nanocomposite design using surface modified silver nanowires decorated on the surface of polyethyleneimine (PEI), a cationic polymer acting as glue

for anchoring nanowires and reduced graphene oxide (rGO). The synthesized nanocomposite was employed as a promising electrode material for immobilization of biomolecules and effective

transportation of electron, in enzymatic biofuel cell (EBFCs) application. The synthesized nanocomposite was confirmed by analytical techniques, for instance, Fourier transform infrared

spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM). The electrochemical behaviour of the nanobioelectrocatalysts rGO-PEI/Frt/GOx,

rGO-PEI/AgNWs/Frt/GOx, and rGO-PEI/Naph-SH/AgNWs/Frt/GOx was determined by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV). The

maximum current density obtained by the modified bioanode was found to be 19.9 mA cm−2 at the limiting glucose concentration of 50 mM in PBS (pH 7.0) as supporting electrolyte at a scan rate

of 100 mVs−1. SIMILAR CONTENT BEING VIEWED BY OTHERS IMPROVING THE POWER PRODUCTION EFFICIENCY OF MICROBIAL FUEL CELL BY USING BIOSYNTHESIZED POLYANALINE COATED FE3O4 AS PENCIL GRAPHITE

ANODE MODIFIER Article Open access 02 January 2025 FABRICATION AND CHARACTERIZATION OF ELECTRICALLY CONDUCTING ELECTROCHEMICALLY SYNTHESIZED POLYPYRROLE-BASED ENZYMATIC BIOFUEL CELL ANODE

WITH BIOCOMPATIBLE REDOX MEDIATOR VITAMIN K3 Article Open access 09 February 2024 APPLICATIONS OF CHITOSAN (CHI)-REDUCED GRAPHENE OXIDE (RGO)-POLYANILINE (PANI) CONDUCTING COMPOSITE

ELECTRODE FOR ENERGY GENERATION IN GLUCOSE BIOFUEL CELL Article Open access 26 June 2020 INTRODUCTION Nanoscience and nanotechnology are gaining increasing interest due to their ability to

tailor diverse nanoscale materials. Interestingly, nanomaterials possess excellent properties such as large surface to volume ratio, high surface reactivity, and different morphologies1,2,3.

These dynamic properties of nanomaterials make them advantageous in any conceivable domain like in the food industry4, electronics5, pharmaceuticals6 and agriculture7. Nanoscale materials

have also been utilized in enzymatic biofuel cells (EBFCs) to improve their performance8. EBFC is one of the reliable energy generation technologies for powering miniaturized bioelectronic

systems. Enzymatic biofuel cells relay on two separate redox reactions coupled with the enzyme functionalized electrode materials linked through an external circuit9. The potential chemical

energy stored in biomolecules is converted into electrical energy by a suitable biocatalyst. The merits of the generation of electrical power via EBFCs are their eco-friendly and feasible

operational conditions, use of renewable physiological fluids (e.g., glucose10, fructose11, etc.) and specificity of biocatalysts. For example, the glucose oxidase (GOx)12 is a

bioelectrocatalyst that specifically catalyzes the most exploited glucose fuel, which is ubiquitous in the living systems. However, the redox-active centre of GOx is deeply seated inside its

protein shell which makes the electron transfer difficult through it13. Two main strategies such as the use of redox active mediator and orientated immobilization of enzymes have been used

to mitigate this problem14,15,16. Furthermore, the capability of nanomaterials such as carbon nanotubes or metal nanoparticles to link directly to the electroactive enzyme is of immense

interest to facilitate the direct electron transfer (DET)17 while in the case of mediated electron transfer (MET), a mediator is employed in between the electrode surface and the enzyme that

acts as an electron relay center by reducing the path length for electron transfer14. Reportedly, a 14-fold increase in catalytic current was obtained by using a free naphthoquinone to a

glucose oxidase (GOx)/multi-walled carbon nanotube (MWCNT)-based anode16. Nowadays, ferritin (Frt) has been employed as an active redox mediator that assists the electron shuttling between

redox active site of GOx and the electrode surface18. The biocompatible nature of Frt makes it more favourable than the other toxic mediators which enhances its utilization in a

glucose-based biofuel cell19. The specificity of the GOx towards the oxidation of glucose leads to the development of the miniaturized membrane-less system20 which makes EBFCs an essential

tool in the field of implantable power sources, self-powered sensors21, and portable electronic devices22. However, despite all the promises, still, there are specific roadblocks such as low

power output and short life span which limit their real-world applications. Over the past few decades, the development of advanced supporting materials is the intellectual strategy in

material research to achieve efficient immobilization of enzymes23. Recent progress in nanoscience and a detailed understanding of the chemistry of nanomaterials ensure cost-efficient

development of nanobiocatalysts24. To date, carbon nanomaterials (e.g., carbon nanotubes, graphene, etc.) have been exploited as a versatile supporting material for the enzyme wiring25.

Currently, researchers are showing great interest in exploiting the intrinsic properties of 2D graphene and its derivatives (graphene oxide, reduced graphene oxide) in various fields such as

nanoelectronics26, drug delivery27, environmental remediation28 and designing electrode materials29, on account of their high mechanical strength, good electrical conductivity, free

electron mobility, high aspect ratio, flexible tunability, and lightweight. The high surface to volume ratio of rGO offers the large accessible surface area for enzyme loading30. To date,

various combinations of rGO with conducting polymers such as PANI-rGO31, polypyrrole-rGO32 have been reported as potential electrode materials. However, very few articles have been published

on polyethyleneimine (PEI) with carbon nanomaterials33 in biofuel cells. However, some applications have been explored in biosensors34, filtration membrane35 and drug delivery36. PEI is a

biocompatible porous cationic polymer that is easily prepared and economically feasible. It is also renowned for its excellent enzyme immobilizing ability. Under normal conditions, it

possesses a positive charge which maintains strong interaction with GOx. Although PEI possesses all these fascinating attributes, still its conductivity is lowe as compared to that of others

conducting polymers such as polypyrrole, polythiophene, and polyaniline which limits its direct application as electrode materials37. To address the above issues, the use of metal

nanoparticles (MNPs) can be a promising strategy to improve the conductivity of PEI. The utilization of surface modified MNPs overcomes the problem of their aggregation and simultaneously

promotes their interaction with the polymer matrix. A variety of MNPs such as gold38, magnetite39 and silver nanoparticles40 have been explored for electrodes modification due to their high

catalytic activity. Similarly, the capability of electron transfer surges by introducing the thiol group on the surface of MNPs41. Furthermore, the morphology of MNPs, their shape, size, and

concentration influence the properties of the nanocomposite. Besides, one-dimensional nanowire has high surface activity due to the very high specific surface area which tremendously

enhances the loading capability of the nanocomposite. Herein, a nanocomposite composed of silver nanowires (AgNWs) modified with naphtalenethiol coupling agent decorated on the surface of

cationic polymer polyethyleneimine (PEI) and the rGO is prepared. Here, PEI acts as glue for anchoring nanowires assisted by thiol linkage of naphthalene thiol. Apart from it,

PEI/AgNWs@Naph-SH acts as a sandwich between rGO sheet and GOx in the developed biocatalyst rGO/PEI/AgNWs@Naph-SH/Frt/GOx. Moreover, modification of AgNWs with a napthalenethiol stabilizing

agent makes a protecting covering around the NWs that prevents them from further ionization. The contribution of each component of the synthesized nanocomposite rGO/PEI/AgNWs@Naph-SH plays a

significant role in improving the stability of the bioanode by establishing (i) hydrophobic interaction between GOx and the PEI/AgNWs@Naph-SH nanocomposite and (ii) π conjugation between

GOx and naphthalenethiol@AgNWs that assist the electron transfer process. In this study, three different types of electrodes; rGO/PEI/Frt/GOx, rGO/PEI/AgNWs/Frt/GOx, and

rGO/PEI/AgNWs@Napth-SH/Frt/GOx have been developed and compared to confirm the contribution of each component in enzyme wiring. MATERIALS AND METHODS The polyethyleneimine (PEI) (average

molecular weight 800 by light scattering), reduced graphene oxide (rGO), napthalenethiol (Naph-SH,99%), silver nitrate (AgNO3), ferritin (10 mg mL−1 in 0.15 M NaCl), glucose oxidase (GOx)

from Aspergillus niger and 2% glutaraldehyde aqueous solution were purchased from the Sigma-Aldrich, India. Sodium chloride (NaCl), potassium bromide (KBr), phosphate buffer saline (PBS, pH

7.0), polyvinylpyrrolidone (PVP, molecular weight 1,300,000) and ethylene glycol (EG) were procured from Alfa Aesar, India. INSTRUMENTS USED The Fourier transform infrared spectroscopy

(FTIR) was carried out with the help of Nicolet iS50 FT-IR instrument operating in the range of 4000–400 cm−1, and X-ray diffraction (XRD) analysis was performed by using Rigaku Smart Lab

X-ray diffractometer, to elucidate the functional groups and the crystalline structure of the nanocomposite, respectively. The morphologies of the synthesized nanocomposites were examined

using scanning electron microscopy (SEM) (JSM, 6510 LV, JEOL, Japan) and transmission electron microscopy (TEM) (TEM 2100, JEOL, Japan) operated at 200 kV on a carbon-coated copper grid.

Moreover, the electrochemical behaviour of the fabricated bioanode was investigated by a three-electrode assembly having glassy carbon electrode as working, a Pt wire as a counter and

Ag/AgCl (3 M KCl) as a reference electrode, coupled with the potentiostat/galvanostat (PGSTAT 302 Autolab, Switzerland). SYNTHESIS OF SILVER NANOWIRES (AGNWS) The synthesis of silver

nanowires (AgNWs) through controlled morphology can be obtained by keeping the molar ratio of [PVP] to [AgNO3] as mentioned elsewhere42. Briefly, PVP (75 mg) was dissolved in 10 mL of EG

with continuous heating and magnetic stirring (550 revolutions per minute) of the solution in an oil bath for 60 minutes at 160 °C temperature. After that, 20 µL of KBr (1.8 mM) and 40 µL of

NaCl (3.5 mM) solutions prepared in EG were added into the above solution followed by stirring and heating of the mixture for 30 minutes. Then, 5.2 ml AgNO3 solution was pipetted out and

continuously added to the above mixture while maintaining the flow rate of 0.2 mL min−1. Following this, the solution was refluxed at 160 °C upto 3 h and the noticeable change in the colour

was observed from yellow to turbid grey which indicates the formation of AgNWs. After cooling, the solution was agitated for 10 minutes in an ultra-sonication bath and centrifuged at 7000 

rpm. Lastly, final pure AgNWs as shown in the SEM micrograph were obtained by decanting the supernatant liquid. SYNTHESIS OF RGO/PEI/AGNWS@NAPH-SH NANOCOMPOSITE Firstly, the 100 mg of rGO

was dispersed in 5 mL double distilled water (DDW) for the proper dispersion of rGO sheets and then 20 mL PEI polymer was added into it; the negatively charged rGO sheets interact with

positively charged PEI resulting in the homogenous suspension. Simultaneously, in a separate beaker, 1 mL of already synthesized AgNWs were ultrasonicated with 2 mg naphthalenthiol coupling

agent and centrifuged for 7 minutes. This resulting mixture was added into above mentioned rGO/PEI homogenous suspension and diluted it with distilled water so that the final concentration

of the solution reached 2.5 mg mL−1. The mixture was ultrasonicated for 10 minutes allowing the reaction to complete. The supernatant was dumped followed by 7 minutes centrifugation and this

step was repeated three times by washing with DDW to ensure the removal of excessive PEI. The same procedure was followed for the synthesis of rGO/PEI/AgNWs without the modification of

AgNWs41. PREPARATION OF RGO/PEI/AGNWS@NAPH-SH NANOCOMPOSITE DISPERSION The consistency of the synthesized (rGO/PEI/AgNWs@Naph-SH) nanocomposite was adjusted by adding double-distilled water

into it and the resultant dispersion was used as catalytic ink. FABRICATION OF BIOANODE The bioanodes rGO/PEI/Frt/GOx, rGO/PEI/AgNWs/Frt/GOx, and rGO/PEI/AgNWs@Naph-SH /Frt/GOx were

developed by the drop-casting method. Before using, a 3 mm diameter glassy carbon electrode (GCE) was cleaned as described elsewhere43. For designing the bioanode, firstly 6 µL of the

synthesized nanocomposite (rGO/PEI/AgNWs@Naph-SH) dispersion was drop-cast on the top of GCE by spreading evenly using micro-pipette and letting it dry for 5–6 hours at room temperature.

After that, 5 µL of Frt solution was applied on the dried GCE and allowed to dry for 30 minutes. Next, 6 µL of GOx solution was loaded on the modified GCE. Thereafter, 1.5 µL of 2% aqueous

solution of glutaraldehyde was coated on the bioanode to crosslink the enzyme. After that, the modified electrode was dipped in DDW for 2 minutes to wipe off the unloaded enzyme and kept the

modified electrode in the refrigerator (4 °C) before conducting the experiment. The remaining electrodes were fabricated similarly. RESULTS AND DISCUSSION FTIR ANALYSIS Figure 1 represents

the FTIR spectra of the AgNWs, rGO and the synthesized rGO/PEI/AgNWs nanocomposite. In Fig. 1(a), the band at 1024 cm−1 attributes to the C-N stretching vibration of PVP whereas the

vibrations around 1636 cm−1 indicate the presence of the C = O group in PVP44. The bands (Fig. 1(b)) at around 1036 and 3434 cm−1 can be assigned to the C-C and O-H stretching vibrations of

rGO, respectively45. The FTIR spectrum of rGO/PEI/AgNWs nanocomposite as shown in Fig. 1(c) displays almost the same vibration peaks as found in the individual spectrum of its components

including a peak at 1656 cm−1 due to N-H bending vibrations of PEI46,47. XRD ANALYSIS The recorded X-ray diffraction patterns of the rGO and AgNWs were compared with as-prepared

rGO/PEI/AgNWs nanocomposite as shown in Fig. 2. The vibrations corresponding to 26° can be attributed to the hexagonal plane (002) of rGO displayed in Fig. 2(a)48. The four sharp diffracting

peaks of as-synthesized AgNWs in Fig. 2(b) were located at 2Ѳ angle of diffraction, for instance, 38.2°, 44.4°, 64.6° and 77.6° which are in agreement with the (111), (200), (220) and (311)

diffractions of face-centered cubic (FCC)42. The diffraction peaks found in the synthesized rGO/PEI/AgNWs nanocomposite (Fig. 2c) represent almost all the diffraction peaks as found in each

component and the amorphous nature appears due to the presence of PEI49. Hence, the XRD pattern also confirms the successful synthesis of the nanocomposite as well as of AgNWs. SEM AND TEM

ANALYSES The morphology of the synthesized AgNWs and the nanocomposite (rGO/PEI/AgNWs@Naph-SH) are represented in the SEM micrographs. Figure 3(a) displays the rod shape, smooth texture and

highly purified uniform nanowires of silver. From this image, it can be concluded that the AgNWs have been synthesized successfully. The SEM micrograph of nanocomposite illustrated in Fig. 

3(b) clearly showed that the rGO sheets are wrapped around the PEI surface. The uniform dispersion of rGO sheets in the polymer matrix occurs due to the covalent interaction between rGO and

PEI. Also, it can be seen in Fig. 3(c) that the rough surface ensures the wiring of GOx over the rGO/PEI/AgNWs@Naph-SH nanocomposite. Moreover, the TEM micrographs at different

magnifications as shown in Fig. 4(a,b) revealed the non-uniform parallel arrays of AgNWs clinging to the surface of PEI and rGO sheets which further display the microenvironment of the

nanocomposite. It could be concluded that AgNWs and rGO sheets act as fillers in a polymer matrix that provides a continuous path for electron transfer. ELECTROCHEMICAL STUDIES The

electrocatalytic performance of the modified electrodes namely, rGO/PEI/Frt/GOx, rGO/PEI/AgNWs/Frt/GOx, and rGO/PEI/AgNWs@Naph-SH/Frt/GOx were examined by cyclic voltammetry (CV). Before the

examination, N2 gas was bubbled into the PBS (pH 7.0) to provide an inert environment. The electrochemical testing of the fabricated electrodes was conducted at the ambient temperature in

0.1 M PBS (pH 7.0) containing 50 mM glucose. In this study, the contribution of pure AgNWs and AgNWs@Naph-SH on the catalytic activity of biocatalyst was evaluated by comparing the CV curves

of fabricated electrodes as shown in Fig. 5. The outcomes that appeared in CV curves illustrate that the current density obtained from the redox reaction of FAD (Flavin adenine

dinucleotide) on the rGO/PEI/AgNWs/Frt/GOx bioanode was considerably higher than the rGO/PEI and rGO/PEI/Frt/GOx. It means that AgNWs favour the shuttling of the electron between the GOx and

electrode surface, whereas the voltammogram of rGO/PEI/AgNWs@Naph-SH/Frt/GOx bioanode displayed further enhancement in the intensity of the catalytic current. This enhancement in the

current density of rGO/PEI/AgNWs@Naph-SH/Frt/GOx bioanode can be attributed to the involvement of Naph-SH, which assists the electron shuttling by forming the Ag-thiol linkage. This linkage

creates a hydrophobic interaction with the GOx that accelerating the electron kinetics of the reaction. Additionally, Naph-SH improved the electron transfer capability of PEI by promoting

the electrical communication between the GOx and rGO sheets via π-π stacking50. Moreover, it can be seen from the figure that no catalytic activity was delivered by voltammogram of GOx due

to leakage or poor immobilization of enzyme over the bare GCE. Furthermore, the biocatalytic activity of the fabricated electrode rGO/PEI/AgNWs@Naph-SH/Frt/GOx was also studied as a function

of scan rate ranging from 20 to 100 mVs−1 as shown in Fig. 6(a). From the figure, it is evident that by increasing the scan rate, the current density of the specified electrode increases

linearly. The noticeable increment in current density is because of the high voltage applying that reduces the diffusion layer formed at the interface and facilitates electron transfer. The

calibration plot of redox peak current Vs scan rate is given in Fig. 6(b). The curve demonstrates that the magnitude of peaks current (anodic and cathodic) rises as the potential scan rate 

increases linearly from 20 to 100 m Vs−1. So, it can be concluded that the catalytic reactions follow surface controlled processes51. One of the valuable information which gives insight

about the kinetics of the modified electrodes is the evaluation of rate constant (ks) using Laviron’s equation52. The ks values of the three developed electrodes were evaluated as, 8.7 s−1,

9.8 s−1, 11.2 s−1 at 100 mVs−1 scan rate. More interestingly, the ks values indicates that the use of AgNWs and AgNWs@Naph-SH contribute to enhancing the electron transfer rate. These

outcomes show a good response as compared to the reported catalyst as shown in Table 153,54,55,56,57,58. Also, the surface concentration of the enzyme immobilized on

rGO/PEI/AgNWs@Naph-SH/Frt/GOx was estimated to be 3.4 × 10−7 mol cm−2 by using Brown-Anson model as given in Eq. (1)43. $${I}_{p}=\frac{{n}^{2}{F}^{2}{I}^{\ast }AV}{4RT}$$ (1) where Ip = 

anodic peak current at 100 mVs−1 scan rate, I* = surface concentration of rGO/PEI/AgNWs@Naph-SH/Frt/GOx to be determined, A = surface area of GC electrode (0.07 cm−2). R (gas constants;

8.314JK−1), T (temperature, 298 K), F (Faraday’s constant; 96485 Cmol−1), n = 2 (number of electron transfer). EIS STUDY Electrical impedance spectroscopy (EIS) was used to characterize the

developed electrodes as shown in Fig. 7. By using Nyquist plots, the charge transfer resistance (Rct) of three fabricated electrodes was measured. Fig. 7 showed that the electrolyte

resistance (Rs) remained the same for all the three developed catalysts. The rGO/PEI/Frt/GOx displays a well-defined semi-circle in the high-frequency region with the interface electron

transfer resistance (Rct) of 430 Ω (curve c). After incorporating the AgNWs, the diameter of the semi-circle reduced corresponding to Rct 410 Ω (curve b). The decrease in Rct ascribes the

highly conducting nanowires of silver, which assist the electron shuttling by forming a continuous phase in the nanocomposite. Subsequently, the decrease in Rct to 260 Ω (curve a) was

obtained for rGO/PEI/AgNWs@Naph-SH/Frt/GOx. It can be due to Ag-thiol linkage which further enhances the flow of electron50. LSV ANALYSIS Linear sweep voltammetry (LSV) was carried out to

optimize the current density by varying the glucose concentration of rGO/PEI/AgNWs@Naph-SH/Frt/GOx bioanode in PBS (pH 7.0). It is clear from Fig. 8(a) that the electrocatalytic current

density enhances dramatically with the rise in glucose concentration from 10 to 50 mM. Upon addition of 60 mM glucose, there is a decrease in the anodic current, suggesting its saturation at

50 mM glucose. From this result, it can be concluded that the rise in current occurred due to the availability of vacant sites on the catalyst surface that are active to bind with substrate

molecules and once all the active sites get filled, the saturation was reached leading to further decrease in current density. Moreover, the respective calibration curve of synthesized

bioanode displays a maximum current density of 19.9 mAcm−2 at the 50 mM glucose concentration in Fig. 8(b). The proposed work is compared with the reported work as given in Table 

218,56,57,58,59. The current density of the proposed electrode is comparable with the electrodes already available. Moreover, the fabricated electrode can be used upto 45 days while keeping

it into the refrigerator at 4 °C when not in use. CONCLUSION In this work, three nano-scaffold rGO/PEI, rGO/PEI/AgNWs, and rGO/PEI/AgNWs@Naph-SH were synthesized. The catalytic activity of

these nano-scaffolds was tested by immobilizing enzyme (GOx) and mediator (Frt). The catalytic performance of the immobilized enzyme was examined by comparing the rGO/PEI/Frt/GOx,

rGO/PEI/AgNWs/Frt/GOx and rGO/PEI/AgNWs@Naph-SH/Frt/GOx bioanodes. The maximum electrocatalytic current density, higher ks 11.2 s−1 and lower charge transfer resistance (260 Ω) were found to

be in the case of rGO/PEI/AgNWs@Naph-SH/Frt/GOx. Thus, our outcomes will likely open the doors for the development of more efficient EBFCs by using PEI-rGO films as the electrode supports.

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of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (D-195-130-1440). The authors, therefore, gratefully acknowledge the DSR technical and financial support.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia Inamuddin * Advanced Functional Materials

Laboratory, Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh, 202 002, India Inamuddin * Department of Chemistry, Faculty of

Science, Aligarh Muslim University, Aligarh, 202002, India Nimra Shakeel Authors * Inamuddin View author publications You can also search for this author inPubMed Google Scholar * Nimra

Shakeel View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS I. and N.S. contributed to ideas, executed all the experiments, analysed and

interpreted the data. Both the authors contributed to the discussion and agreed to submit the manuscript. CORRESPONDING AUTHOR Correspondence to Inamuddin. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

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THIS ARTICLE CITE THIS ARTICLE Inamuddin, Shakeel, N. Optimization of rGO-PEI/Naph-SH/AgNWs/Frt/GOx nanocomposite anode for biofuel cell applications. _Sci Rep_ 10, 8919 (2020).

https://doi.org/10.1038/s41598-020-65712-8 Download citation * Received: 30 March 2020 * Accepted: 08 May 2020 * Published: 02 June 2020 * DOI: https://doi.org/10.1038/s41598-020-65712-8

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