A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode

A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode Gamze Emir, Yusuf Dilgin, Samet Sahin, Cahit Akgül This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4376605/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract Enzymatic biofuel cells (EBFC) are promising sources of green energy owing to the benefits of using renewable biofuels, eco-friendly biocatalysts, and moderate operating conditions. In this study, a simple and effective EBFC was presented using an enzymatic composite material-based anode and a nonenzymatic bimetallic nanoparticle-based cathode, respectively. The anode was constructed from a glassy carbon electrode (GCE) modified with a multi-walled carbon nanotube (MWCNT) and ferrocene (Fc) as a conductive layer coupled with the enzyme glucose oxidase (GOx) as a sensitive detection layer for glucose. A chitosan layer was also applied to the electrode as a protective layer to complete the composite anode. On the other hand, the cathode consisted of a disposable pencil graphite electrode (PGE) modified with platinum-palladium bimetallic nanoparticles (Nps) which exhibit excellent conductivity and electron transfer rate for the oxygen reduction reaction. The EBFC consisting of MWCNT-Fc-GOx/GCE anode and Pt-PdNps/PGE cathode exhibits an open circuit potential of 285 mV and a maximum power density of 32.25 µW cm − 2 under optimized conditions. The results show that the proposed EBFC is a promising candidate for detecting glucose while harvesting power from artificial serum samples. Enzymatic biofuel cell (EBFC) Glucose Oxidase (GOx) Nanozyme Self-Powered Glucose Sensor (SPGS) Ferrocene (Fc) Bimetallic Nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Biofuel cells (BFCs) are electrochemical devices that generate electrical energy using organic molecules produced during metabolic processes as a fuel and a renewable biological catalyst. They are considered environmentally friendly and sustainable energy options [ 1 – 5 ]. Two main BFCs have become prominent: enzymatic biofuel cells (EBFCs) that use purified oxidoreductase enzymes and microbial biofuel cells (MBFCs) that use microorganisms [ 3 – 6 ]. EBFCs are attractive devices that directly convert the chemical energy stored in the fuel into electricity using enzymes as biocatalysts. These types of fuel cells are commonly employed in biosensors and bioelectronic devices. EBFCs transform enzymatic chemical reactivity into electrical power output by oxidizing fuel at the anode and reducing an oxidant at the cathode [ 4 – 6 ]. EFBCs are known as renewable and environmentally friendly technologies due to their remarkable properties such as easy miniaturization, portability, working conditions around room temperatures and physiological pH. Furthermore, EBFCs offer advantages such as high conversion efficiency and versatility as they can generate electrical energy from various organic substrates [ 5 – 7 ]. Separating the anodic and cathodic parts is unnecessary thanks to the highly selective enzymes. This enables the development of miniature and membrane-less EBFCs implanted in the human body to potentially power medical devices [ 5 , 8 – 10 ]. However, current approaches face several challenges, such as the need for specific pH and temperature conditions, difficulties in stabilizing enzymes to the electrode surfaces and inadequacies in electron transfer rate between the electrodes [ 9 ]. Numerous techniques have been developed to improve the enzyme immobilization, power output and stability of EBFCs. Various enzyme immobilization techniques, including direct immobilization, cross-linking with bifunctional linker molecules and self-assembled monolayers, have already been applied to EBFCs [ 10 , 11 ]. Various electrode surface modifications were performed using novel materials to promote electron transfer between the redox enzyme and electrode [ 10 – 13 ]. For this reason, enzyme electrodes have been prepared using a variety of materials, like gold nanoparticles [ 14 – 16 ], carbon nanotubes, redox polymers, metal complexes [ 17 , 18 ], or electron transfer mediators, which include osmium (Os), benzoquinone, polyvinyl ferrocene, ferrocene (Fc) and its derivatives [ 19 , 21 ] Redox mediators are widely used in biosensors, energy conversion, bio-electrocatalysis and storage technologies. Ferrocene (Fc) and its derivatives are notable among all the mediators used due to their nontoxicity to the human body and solubility in various solvents, including water and ethanol [ 3 , 21 ] Carbon nanotubes (CNTs) have also been used to improve the properties of Fc-modified electrodes because of their unique biocompatibility properties and excellent electrical conductivity [ 3 , 17 , 21 ]. Improving the oxygen reduction reaction (ORR) activity is essential for fuel cell development [ 22 ]. The glucose/O 2 biofuel cell, with a glucose oxidizing anode and an O 2 -reducing cathode, could become an in vivo electricity source to power integrated medical devices. Recent developments in nanotechnology have led to the designing of new artificial enzymes called nanozymes. Nanozymes have numerous advantages, including diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, ease of surface tunability and biocompatibility. These advantages have allowed their integration into a wide range of biosensing applications [ 23 , 26 ]. Several biosensing systems have been developed using metal, metal oxide and metal-organic framework-based nanozymes [ 23 – 26 ]. In this work, we designed a simple EBFC configuration operating in ambient and physiological conditions containing enzymatic composite material-based anode and nonenzymatic bimetallic nanoparticle-based cathode. Glucose was selected as the substrate due to its suitability as a biological fuel and its prevalence in various biological substrates such as blood and tears. In our system, glucose is oxidized by glucose oxidase (GOx) at the anode and oxygen is reduced by bimetallic nanoparticle composite material at the cathode. The anode consists of a GOx immobilized on a glassy carbon electrode (GCE) modified with an MWCNT and Fc. On the other hand, the cathode consisted of a pencil graphite electrode modified with platinum (Pt) and palladium (Pd) metal nanoparticles (MNps). Pencil graphite electrode (PGE) was preferred as an electrode material at the cathode because of its advantages, such as electrochemical activity, commercial accessibility, good mechanical strength, disposability, low cost, and simple modification procedure [ 26 , 27 ]. This novel design of EBFC has produced significant electrical power that can be used for various applications, including glucose detection or powering medical devices. 2. Materials and Methods 2.1. Reagents Analytical-grade chemicals were used without further purification. GOx (from Aspergillus niger type 77 units/mg) was purchased from Sigma Aldrich. Stock solutions of GOx were prepared using 0.1 M phosphate buffer solution (PBS) with pH 7.0, and stored at -20 ◦ C until used. Multi-walled carbon nanotubes, ferrocene, palladium (II) chloride (PdCl 2 ), and chloroplatinic acid (H 2 PtCl 6 ) solution 8% wt. in H 2 O was supplied from Sigma Aldrich. Chemicals such as potassium phosphate dibasic (Na 2 HPO 4 .2H 2 O), potassium phosphate monobasic (NaH 2 PO 4 .2H 2 O), potassium chloride (KCl), sodium hydroxide (NaOH), chitosan (medium molecular weight), D-(+)-glucose (≥ 99.5%) were purchased from Merck. Potassium ferricyanide (K 3 [Fe(CN) 6 ].3H 2 O) and potassium ferrocyanide (K 4 [Fe(CN) 6 ].3H 2 O) were purchased from Sigma Aldrich. All solutions were prepared with ultrapure water using the Elga Option Q7B water purification system (18.2 µW cm -1 ). 0.10 M KCl containing 0.1 M PBS with pH 7.0 was used as an electrolyte to characterize electrodes and biofuel cell tests. 2.2. Apparatus and Electrochemical Measurements All electrochemical measurements, including electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), linear sweep voltammetry (LSV), amperometry and open circuit potential (OCP) tests were run using a Metrohm model Autolab PGSTAT 128N Potentiostat/Galvanostat. Before carrying out the electrochemical tests and between each successive addition of glucose, it was ensured that the solutions were purged with either air or argon. The stock glucose solutions were allowed to mutarotate for at least 24 hours before use and then stored in a refrigerator at 4°C. CV and amperometric techniques individually characterized the electrodes employed as anodes and cathodes. In these cases, a three-electrode cell constituted by each unmodified or modified electrode as a working electrode, an Ag/AgCl (KCl, 3 M) as a reference electrode, and a platinum wire as an auxiliary electrode was used. 2.3. Fabrication of the Electrodes 2.3.1. Preparation of Anode Electrodes (MWCNT-Fc-GOx/GCE) Before a modification step, GCEs were polished with different grain sizes (1 µm, 0.3 µm, and 0.05 µm, respectively) alumina on a polishing cloth. Then, GCE was sonicated in ethanol and ultrapure water and dried with an IR lamp. Then, the dried electrodes were placed in an electrochemical cell, and a preconditioning step of 20 scans at 50 mV s − 1 between − 0.4 V and 0.4 V (vs. Ag/Ag + ) was applied in 0.1 M KCl with the CV method. After the preconditioning step, the electrodes were washed with de-ionized water and dried in front of IR lamb for 5 minutes. After the cleaning procedure of GCE, 25 mM Fc containing 20 mg/mL MWCNT solution, was prepared and sonicated for 15 minutes in an ultrasonic bath. Then, 10.0 µL from that solution was added onto the GCE surface and dried with IR lamb for 5 minutes in every 1.0 mL addition. 20 mg/mL GOx mixed in 0.10 M PBS with pH 7.0, was then dropped on the surface of MWCNT-Fc-GCE, and dried for 2 hours at the room temperature. The preparation of the enzyme electrode is finalized by the addition of the % 0.5 chitosan solution in acetic acid. The enzyme electrodes were stored at + 4°C when not in use. 2.3.2. Preparation of cathode electrodes (PtNps/PGE, PdNps/PGE and Pt-PdNps/PGE) Platinum nanoparticles (PtNps) have been efficiently deposited onto the surface of PGE using an electrochemical method. It was achieved by recording cyclic voltammograms in a 5.0 mM solution of H 2 PtCl 6 containing 0.10 M KCl from 0.8 V to -0.8 V at a scan rate of 50 mVs − 1 for 30 cycles. (Fig. S1 A). electrodeposition mechanism of PtCl 6 2− was reported by our previous study [ 27 ]. Palladium nanoparticles (PdNps) were also efficiently deposited onto the surface of PGE using an electrochemical method [ 28 ]. Cyclic voltammograms were recorded in a supporting electrolyte of 0.10 M KCl and 5.0 mM PdCl 2 at a scan rate of 25 mVs − 1 for 30 cycles within a potential range of 0.40 V to -0.25 V (Fig. S1 B). For the PtPd-Nps electrodes, 5.0 mM of H 2 PtCl 6 and 5.0 mM of PdCl 2 containing 0.1 M KCl solutions were used. This is also achieved by recording CV under the same conditions as PtNps and PdNps deposition, respectively (Fig. S1 C). 2.4. Characterization of Electrodes In this study, SEM and EDX images of both unmodified and modified PGE electrodes were taken to provide an accurate analysis of the surface changes resulting from the electrochemical processes. Scanning electron microscopy (SEM-EDX) images was taken at Experimental Science Application and Research Center (DEFAM) of Manisa Celal Bayar University with a device of Zeiss, Gemini 500 model. Also, the electrodes were electrochemically characterized using CV and EIS techniques, which were recorded in a solution containing 5.0 mM Fe(CN) 6 3−/4− + 0.10 M KCl. 2.5. Fuel Cell Tests and Real Sample Analysis The fully assembled biofuel cells were characterized by recording LSV using MWCNT-Fc-GOx/GCE as anode and Pt-PdNps/PGE as a cathode in oxygen-saturated 0.1 M PBS at pH 7.0 with different glucose concentrations. LSV was performed at 1.0 mV s − 1 from the OCP of the cell to 0 V to evaluate the performance of the designed EBFCs. The current and power of the fuel cell were calculated using Ohm's Law (V = I × R and P = I × V). The output density and current density were calculated from the apparent surface area of the anode. All the measurements were carried out at 37 ºC. The constructed glucose-based self-powered biofuel cell was tested using a sample of artificial blood serum containing 4.77 mM glucose. The performance of the designed EBFCs in real samples was evaluated using LSV. 3. Results and Discussions 3.1. Characterization of Nanozyme-Based Cathode (Pt-Pd-Nps/PGE) and Its Electrochemical Response to ORR In the first stage of the studies, the characterization of the cathode was carried out, and its electrocatalytic activity with the reduction of O 2 was examined. It is well known that biocathodes consisting of enzymes such as bilirubin oxidase and laccase have been extensively used in developing EFCs due to their direct electron transfer ability and selectivity [ 26 , 29 – 31 ]. These properties allow them to operate without a separation membrane to prevent cross-reactions at the electrodes [ 26 ]. Enzyme-mimicking nanomaterials called nanozymes have recently been introduced to biosensor and fuel cell studies [ 32 – 34 ]. In this study, PGEs modified with Pt-Pd bimetallic nanoparticles used as nanozyme have been proposed as a new cathode material in biofuel cell studies for the first time. First, PtNps, PdNps and Pt-PdNps were modified on PGE using electrochemical methods. SEM images and EDX spectra were then recorded for unmodified and modified PGEs. SEM images (Fig. 1 ) show that the layered structures of unmodified PGE are homogeneously covered with nanoparticles obtained from monometal and bimetal deposition. According to EDX results (Fig. S2), it is expected to see %76.93 C, %17.68 O, %0.68 Mg, %1.53 Al, % 0.25 Ca, %2 Fe and %2.75 Si in unmodified PGE because pencil leads consist of clay and graphite [ 35 ]. In addition, 13.38% Pt in PtNps, 8.52% Pd in PdNps, and 4.27% Pt and 16.47% Pd in Pt-PdNps were found, which reflects that monometallic and bimetallic nanoparticles were successfully attached to the PGE surface. EIS is widely used to understand electrode surface changes and resistance properties. The EIS curve consists of two main parts: the semicircle and the linear portion. These parts provide detailed information on the impedance changes of an electrode surface, which is valuable for understanding modifications of electrode surfaces and their use in electrochemical sensing applications [ 36 ]. The linear portion of impedance spectra corresponds to the processes of electron transfer that are limited by diffusion. The semicircle is closely related to high frequencies, and its diameter provides information about the charge transfer resistance (R ct ), which controls the electron transfer kinetics of the redox probe at the electrode's surface [ 36 ]. Thus, unmodified and modified PGEs were also electrochemically characterized by evaluating their CVs and EIS to understand electrode surface changes and surface resistance properties. EIS spectra of unmodified Pt-Nps/PGE, Pd-Nps/PGE, and PtPd-NPs/PGE were recorded in 10.0 mM [Fe(CN) 6 ] 3-/4- containing 0.10 M KCl. The R ct values obtained from Nyquist curves were obtained as 110, 37, 18 and 8 ohms for unmodified PGE, Pd-Nps/PGE, Pt-Nps/PGE, and PtPd-NPs/PGE, respectively (Fig. S3A). This is attributed to the fact that the metal nanoparticles speed up the transfer of electrons on the surface of the PGE. CVs recorded in the redox probe for each electrode agree well with the EIS measurements. It can be seen from CVs that the highest peak currents of the redox probe were obtained from Pt-PdNps/PGE, while the lowest peak currents were obtained from unmodified PGE (Fig. S3B). To compare the electrocatalytic activities of the electrodes (bimetallic NPs-modified PGE), ORR was performed by using unmodified PGE and monometallic modified PGEs (PtNps/PGE and PdNps/PGE) together with Pt-PdNps/PGE as cathodes. Figure 2 shows the CVs and LSVs of these electrodes in the argon and oxygen-saturated 0.1 M KCl containing 0.1 M PBS at pH 7.0. It can be clearly seen that a remarkable enhancement in the electrocatalytic reduction of O 2 was observed at Pd-PtNps/PGE compared to unmodified PGE and the monometallic NPs-modified electrode. On the other hand, ORR has a high overpotential at unmodified PGE, and the current starting at 0 mV did not take the form of a peak but gradually increased by shifting potential in a more negative direction. ORR current of PtNps/PGE, PdNps/PGE, and Pt-PdNps/PGE were found to be 280, 340, and 570 µA at -50, + 50, and + 90 mV vs. Ag/AgCl, respectively. It was concluded that Pt-PdNps/PGE is the most suitable electrode for the cathode in glucose/O 2 type EBFC because bimetallic nanoparticle-modified PGE has the highest peak current at a more positive voltage in the reduction potential compared to monometallic-modified PGEs. 3.2. Characterization and Electrochemical Behavior of Bioanode (MWCNT-Fc-GOx/GCE) Direct electron transfer becomes difficult for GOx since the electroactive group is in the inner parts of the enzyme [ 3 ]. To overcome this situation, electrodes are modified with redox mediators such as ferrocene derivatives and osmium redox polymers [ 17 , 19 , 21 ]. Redox mediators facilitate electron transfer rates between substrate and electrode, resulting in an enhancement in electron transfer at the bioanaode surface and in output power. In this study, ferrocene was immobilized with GOx onto MWCNT/GCE to obtain an effective bioanode. To obtain the optimum MWCNT-Fc-GOx/GCE, which gives the most effective response to glucose oxidation, the GCE surface was optimized by adjusting the different concentrations of MWCNT, Fc and GOx during the electrode preparation step. To determine the optimum electrode composition, CVs of each electrode prepared with different ratios of modification reagents were recorded in the absence and presence of 5.0 mM glucose. Fig. S4. shows the curves of the concentration of modification reagents versus the electrocatalytic oxidation peak current of glucose. The maximum peak current for the electrocatalytic oxidation of glucose was achieved when MWCNT, Fc, and GOx (concentrations of 5.0 mg/mL, 25.0 mM, and 20.0 mg/mL, respectively) were immobilized at GCE surface. The obtained optimum MWCNT-Fc-GOx/GCE was characterized by recording EIS and CVs in the 5.0 mM Fe(CN) 6 4-/3- redox probe containing 0.10 M KCl. The Nyquist curves were used to estimate the R ct , which was then used to evaluate the insulating properties of the electrode/electrolyte interface. Fig. S5A shows that the R ct values were found to be 1610 (Fig. S5A/c), 140 (Fig. S5A/b), and 580 (Fig. S5A/c) ohms for unmodified GCE, MWCNT-Fc-GCE and MWCNT-Fc-GOx-GCE, respectively. R ct value of unmodified GCE (1610 ohm) was remarkably decreased after the modification of both MWCNT and Fc (140 ohm) due to high surface area and conductivity of MWCNT and electrostatic interaction between positively charged Fc and negatively charged redox probe. These results indicate that the conductivity of the MWCNT-Fc/GCE, which is directly related to its electron transfer ability, is higher than that of the unmodified GCE. When the surface of MWCNT-Fc/GCE is immobilized with GOx, R ct is increased to 580 ohms due to steric hindrance between the redox probe and large GOx. In addition, there is good agreement between the CV results given in Fig. S5B and the EIS measurements. CVs from Fig S5B show that the highest peak currents of the redox probe were obtained from MWCNT-Fc/GCE, while the lowest peak currents were obtained from MWCNT-Fc-GOx/GCE. The information obtained from the voltammograms and impedance spectra indicates that MWCNT and Fc play an important role in increasing the electron transfer and conductivity properties between GCE and the Fe(CN) 6 3-/4- redox probe. Finally, the modification procedure with MWCNT-Fc and GOx was successful. GOx is often used as a biocatalyst to produce biosensors and EBFCs. To evaluate the efficacy of MWCNT-Fc-GOx/GCE in catalyzing glucose oxidation, a series of comparative CVs were recorded in the absence and presence of glucose in a 0.1 M PBS with pH 7.0 containing 0.1 M KCl at a scan rate of 20 mVs -1 . It is expected that CV recorded by an unmodified GCE showed no anodic peaks attributed to glucose oxidation (Fig. 3 A). Figure 3 B/a shows that the response of MWCNT-Fc-GOx/GCE in the absence of glucose. A well-defined redox couple with a formal potential of 300 mV indicates high activity and shows a characteristic, reversible redox peak behavior of Fc, in which a single electron transfer is involved for the electro-oxidation peak current of Fc. Also, electrocatalytic oxidation of glucose was observed at the GOx immobilized on the MWCNT-Fc/GCE surface (Fig. 3 B/b). The anodic peak of the MWCNT-Fc-GOx/GCE remarkably increased in the presence of 10 mM of glucose, while in the presence of the same glucose concentration, the cathodic peaks slightly decreased (Fig. 3 B/b). Therefore, the significant increase in the anodic peak of the modified electrodes when exposed to glucose demonstrates the excellent oxidative electrocatalytic activity of the GOx with electrocatalyst (Fc) towards glucose. Considering that the GCE area, which is 0.07 cm², results in a high current density, fast charge transfer on the GCE proves its suitability for electrode modification with GOx, Fc, and MWCNT. CVs of MWCNT-Fc-GOx/GCE were also measured at different glucose concentrations ranging from 0 to 20 mM (Fig. 3 C). The electrodes exhibited a higher oxidative current response with increasing glucose concentrations. They showed the same onset potential (approximately − 0.1 V vs. Ag/Ag + ) in the CV test, which is dependent on the activation of the glucose oxidation reaction. The successful immobilization of enzymes on the electrode surface and electron transfer between the enzyme and electrode via Fc mediator is suggested to have been achieved for Fc-MWCNTs modified electrodes. The electrocatalytic glucose oxidation at the enzymatic electrode (MWCNT-Fc-GOx/GCE) was also investigated by chronoamperometry (CA). To assess the effect of applied potential on the response to glucose, CAs were recorded for MWCNT-Fc-GOx/GCE at various potentials ranging from 0.20 to 0.5 V. This was achieved by adding 1 mM glucose successively into a continuously stirred 10 mL solution of 0.10 M pH 7.0 PBS containing 0.1 M KCl. Results show that maximum peak current was obtained when the applied potential was fixed 0.3 V vs Ag/AgCl. CAs of MWCNT-GOx/GCE and MWCNT-Fc-GOx/GCE at 0.30 V were recorded based on the successive addition of glucose in the range from 1 to 10 mM. The CAs in Fig. 4 A indicate a steady-state current was recorded within approximately 5 seconds of adding glucose. Additionally, it can be observed that these steady-state currents increased as the glucose concentration increased. The MWCNT-GOx/GCE (as shown in Fig. 4 A, red line) does not respond to glucose. However, the MWCNT-Fc-GOx/GCE electrode responds well to glucose. The signal recorded by the modified GCE (Fig. 4 , black line) was higher than that of the MWCNT-GOx/GCE. The calibration plot suggests a linear relationship between current and glucose concentration, which varied from 1 to 10 mM during the study of MWCNT-Fc-GOx/GCE (Fig. 4 B). To calculate the limit of detection (LOD), limit of quantification (LOQ) and sensitivity values of the proposed sensor, the lowest concentration that gives a amperometric response was recorded ten times. The LOD and LOQ were calculated according to the equation of 3xSD/m and 10xSD/m,(“SD”: the standard deviation for peak currents and “m”: the slope calculated from the calibration curve) and determined as 0.261 mM and 0.87 mM, respectively. Also sensitivity of the proposed sensor were calculated 25.71 µA/mM. During the final step of anode characterization, the influence of potentially interfering compounds, such as other monosaccharides (galactose, mannose, fructose, maltose), disaccharides (sucrose), uric acid (UA), dopamine (DA), and ascorbic acid (AA), on the response to glucose was examined at MWCNT-Fc-GOx/GCE. For this purpose, the steady-state background current was stabilized under the optimized conditions. The CAs of MWCNT-Fc-GOx/GCE were recorded by adding known volumes of stock solutions of glucose and interfering compounds to an electrochemical cell. The concentrations of glucose and each interfering compound were fixed at the same concentration. Table S1 demonstrates the response of interference molecules toward glucose. The results show that the designed anode electrode was minimally affected by fructose, sucrose, maltose, mannose, galactose, glutamic acid, lactose, and sodium lactate while highly affected by UA, AA, and DA. It is well known that these substances exhibit positive interference effects by oxidizing at high anodic potentials and enhancing the glucose biosensor's signal. Our previous studies showed that a pre-oxidant (NaBiO 3 ) was successfully used to minimize the interference of these substances [ 37 ]. Thus, solutions, including glucose and interference substances, were passed from NaBiO 3 filled in an injector several times. After that, the interference effects of UA, DA, and AA were significantly reduced by their pre-oxidation with NaBiO 3 , while glucose was not oxidized by NaBiO 3 . The results indicate that MWCNT-Fc-GOx/GCE demonstrates a high selectivity towards glucose. 3.3. Fuel Cell Performance and Real Sample Analysis Glucose was chosen as a biofuel model in our BFC development because it is one of the significant components of human biofluids, including sweat. The fuel cell performance of the system was evaluated by monitoring power output and current density dependent on OCP in air-saturated PBS containing 0.10 M KCl and various concentrations of glucose in the range from 1 to 10 mM. The maximum current density of 460 µA/cm 2 and a maximum power output of 32.05 µW/cm 2 were obtained at a voltage of 0.285 V (Fig. 5 ). The high current and power density were achieved due to the customized composite electrodes in both the anode and cathode. These electrodes possess desirable electrical conductivity and electrocatalytic properties. The hybrid MWCNT-Fc enables an efficient transfer of electrons from the anode to the cathode, significantly increasing the power output. Figure 5 B demonstrates a positive correlation between glucose concentration and power density within the 0–10 mM glucose range. The response generated by the BFC was observed with successive additions of glucose in the range of 0–10 mM. The calibration plot for the self-generated current signal demonstrates linearity up to 10 mM glucose. This observation provides strong evidence for the ability of the BFC to detect glucose levels in this range. Table 1 . compares the performance of the designed glucose-based EBFC with the previously reported EBFCs. As can be seen from Table 1 , the proposed glucose-based EBFC demonstrates better performance than some other previously reported EBFCs in terms of power density values. Furthermore, the designed electrode for the cathode has never been used previously for ORR. PGEs have several benefits, including disposability, affordability, mechanical rigidity, simplicity, commercial availability, and ease of surface modification [ 38 ]. Also, nanozymes have many advantages such as d iverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, ease of surface tunability and biocompatibility. Therefore, our cathode design brings the advantages of PGE and nanozymes together. The mechanism of Pt-Pd-NpsPGE boosting the electrochemical reduction of oxygen can be attributed to the synergistic effect of Pt and Pd nanoparticles, which makes oxygen reduction at the cathode surface easier. Additionally, the cathode electrode can reduce oxygen to water without the need for enzymes such as BOD and Lc. The investigation was conducted to determine the performance of the designed EBFC with artificial serum samples. To attain the desired outcome, the glucose levels in artificial serum samples were measured by the LSV method, and the voltammograms for each sample were recorded under optimized conditions. At 0.280 V, a maximum current density of 290 µA/cm 2 and a maximum power output of 11.35 µW/cm 2 was achieved using 10 mM glucose (Fig. S6). This study has shown that designed EBFC can be effectively used for glucose determination in real and artificial samples and suggests their potential as simple to configure energy-sustaining and non-invasive glucose monitoring devices. Table 1 Comparison of the analytical performance of proposed self-powered EBFC with some previously reported EBFCs in the literature. BFC/analyte Anode Cathode OCP (mV) Power Density (mW/cm 2 ) Current Density (mA/cm 2 ) Reference Glucose/O 2 GDH/poly(TBO)/GCE GOx/HRP/MWCNT/GE 650.0 31.3 165.9 [ 39 ] Glucose/O 2 GRE/5AP/GOx GRE/HRP/GOx 450.0 3.5 22.0 [ 40 ] Glucose/O 2 PGE/MWCNT/GOx PGE/MWCNT/laccase 433.0 18.0 60.0 [ 41 ] Glucose/O 2 PPD/(AuNPs)PPCA‒GOx GRE/PB-PPCA/PPCA‒GOx 640.0 10.94 60.52 [ 5 ] Glucose/O 2 GDH-MB GOx-hemin 500.0 18.0 230.0 [ 42 ] Glucose/O 2 PPy–GOD–CNT PPy–CNT–laccase - 37.4 - [ 43 ] Glucose/O 2 MWCNT-PCA/Ca 2+ /GOx rGC 140.0 6.25 - [ 44 ] Glucose/O 2 Fc-Go/GC BOD-ABTS 2− /fBP 200.0 26 - [ 45 ] Glucose/O 2 MWCNT-Fc-GOx/GCE PtPd-Nps/PGE 285.0 32.05 460.0 In this work (rGC: Reduced graphene oxide-ceria, MWCNT-PCA: Multiwalled carbon nanotube-pyrene carboxylic acid, Fc: Ferrocene, GC: Glassy Carbon Electrode, BOD: Bilirubin Oxidase, ABTS: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt, fBP: functionalized bucky paper, Pt-Pd-Nps: Pt-Pd Nanoparticles, PPy: Poly-Pyrrole, Box: Bilirubin Oxidase, GDH: Glucose Dehydrogenase, MB: Methylene blue, PPD: poly(1,10-phenanthroline-5,6-dione), PB: Prussian blue, PPCA: poly(pyrrole-2-carboxylic acid), MWCNT: Multi-walled carbon nanotube, HRP: Horseradish peroxidase) 4. Conclusion Combining bimetallic and enzymatic catalysts has been suggested as a unique approach to moderate-condition glucose oxidation. This study presents the first evaluation of PGE modified with Pt-Pd bimetallic nanoparticles for oxygen reduction at the cathode. Furthermore, the membraneless design of our EBFC presents another advantage for the developed system. When glucose concentration is 10 mM or higher, the EBFC exceeds a maximal registered open-circuit voltage of 0.285 V. The maximal power density was calculated to be 32.05 µW/cm 2 when the cell voltage was 0.285 V and [glucose] was 10 mM. This could be attributed to the enhanced energetic efficiency due to the use of Fc serving as a mediator for GOx and Pt-Pd bimetallic Nps for ORR. Also, MWCNT can significantly enhance the efficacy of EBFCs by increasing the effective electrode area. Therefore, the EBFC system proposed here may open the possibility of developing more effective energy conversion and applicability to real samples. Declarations Ethical Approval: Not applicable. Consent to Participate: Not applicable. Consent for Publication : We declare that the information in this manuscript has not been published elsewhere nor is it under consideration by any other journal. Furthermore, it is the consensus of all authors to submit this manuscript for possible publication in ABB. Author Contributions: Gamze Emir: Investigation, methodology, analysis and writing original draft; Samet Şahin: Editing, review and supervision; Yusuf Dilgin: Editing, review and supervision; Cahit Akgül: Writing, review & editing, conceptualization, supervision. Funding: We appreciate the Scientific Research Projects Coordination Unit of Çanakkale Onsekiz Mart University (FDK-2022-4073) for financial support. This study was produced as a part of the PhD thesis of Gamze Emir GUNAY. 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Supplementary Files SupplementaryFiles.docx Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Reviewers agreed at journal 19 Jun, 2024 Reviewers invited by journal 19 May, 2024 Editor invited by journal 08 May, 2024 First submitted to journal 07 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4376605","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304272186,"identity":"7bedf95d-b9e6-40e7-83e7-c8a14601501b","order_by":0,"name":"Gamze Emir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDADNgbmA1BmAtFa2GBKgfQB3AqRAY8BcVrk3U8nf/jAYBPNJ33m44ePOYcZ+NlzDJg/7sGtxfBM7gbDGQxpuW18uZslZ247zCDZ88aA4cAzPFoacjck8zAczm3j4d3GzAvUYnAjB6gFj8sM+99uOPyH4T9QC88zsBZ7QlrkJXI3NgO9C9LCBrFFgoAWA4m3mxl7DJKBWtiMgX5J55E486zgwBl8tvTnbv7wo8Iud34P88MPH7dZy/G3J298UIHPFrCcAUKAB0Tg0QC0pQGf7CgYBaNgFIwCEAAADCdSBMrq7aMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3976-2243","institution":"Çanakkale Onsekiz Mart Üniversitesi: Canakkale Onsekiz Mart Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Gamze","middleName":"","lastName":"Emir","suffix":""},{"id":304272187,"identity":"ee311f43-4d5f-4198-bef5-5f48f343891f","order_by":1,"name":"Yusuf Dilgin","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart Üniversites - Terzioğlu Kampüsü: Canakkale Onsekiz Mart Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Yusuf","middleName":"","lastName":"Dilgin","suffix":""},{"id":304272188,"identity":"f7b0ae14-fc0c-4749-93da-1ccb20d21b53","order_by":2,"name":"Samet Sahin","email":"","orcid":"","institution":"Lancaster University","correspondingAuthor":false,"prefix":"","firstName":"Samet","middleName":"","lastName":"Sahin","suffix":""},{"id":304272189,"identity":"8bba4e03-2d06-40dd-99a2-597812552f53","order_by":3,"name":"Cahit Akgül","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart Üniversites - Terzioğlu Kampüsü: Canakkale Onsekiz Mart Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Cahit","middleName":"","lastName":"Akgül","suffix":""}],"badges":[],"createdAt":"2024-05-06 11:55:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4376605/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4376605/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-024-05068-1","type":"published","date":"2024-09-27T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57417306,"identity":"d0987efd-eca7-40c1-a731-ae8ec78fce6a","added_by":"auto","created_at":"2024-05-30 11:57:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":519865,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of \u003cstrong\u003eA)\u003c/strong\u003e unmodified PGE, \u003cstrong\u003eB) \u003c/strong\u003ePt/KGE \u003cstrong\u003eC) \u003c/strong\u003ePd/KGE and \u003cstrong\u003eD) \u003c/strong\u003ePtPd/KGE electrodes\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/85dde857ff64ce04513b30f3.png"},{"id":57417308,"identity":"d3826e1b-7cec-44b3-90b5-537fc41f956b","added_by":"auto","created_at":"2024-05-30 11:57:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217127,"visible":true,"origin":"","legend":"\u003cp\u003eCVs and LSVs of unmodified/PGE (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e), Pd-Nps/PGE (\u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e), Pt-Nps/PGE (\u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e), and Pt-Pd-Nps/PGE (\u003cstrong\u003eG\u003c/strong\u003eand \u003cstrong\u003eH\u003c/strong\u003e) recorded in the supporting electrolyte with 0.1 M PBS at pH 7.0 saturated with argon (black) and oxygen (red). (Scan rate: 25 mV/s)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/bd49eebe7ac88f8ffb664b6a.png"},{"id":57417755,"identity":"ba77855b-7aec-4d0c-a78b-cf16a449292b","added_by":"auto","created_at":"2024-05-30 12:05:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72512,"visible":true,"origin":"","legend":"\u003cp\u003eCVs of: \u003cstrong\u003eA)\u003c/strong\u003e MWCNT-Gox/GCE and \u003cstrong\u003eB)\u003c/strong\u003eMWCNT-Fc-Gox/GCE in the absence (a), presence (b) of 5.0 mM glucose and \u003cstrong\u003eC)\u003c/strong\u003e MWCNT-Fc-Gox/GCE electrode in different glucose concentrations in 0.1 M pH 7.0 PBS containing 0.1 M KCl at a scan rate of 20 mV/s.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/6491014bfba0b1ad6d79a52d.png"},{"id":57417307,"identity":"0f8880c6-a4ae-41eb-8422-1d2607a0c416","added_by":"auto","created_at":"2024-05-30 11:57:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72429,"visible":true,"origin":"","legend":"\u003cp\u003eChrono-amperomograms of \u003cstrong\u003eA)\u003c/strong\u003e MWCNT-Gox/GCE and MWCNT-Fc-Gox/GCE (black line) recorded by the successive addition of 1.0 mM glucose into continuously stirred (at 100 rpm) 10 mL supporting electrolyte (0.10 M of PBS at pH 7.0) at E\u003csub\u003ebias\u003c/sub\u003e of 0.30 V registered at 0.30 V, \u003cstrong\u003eB)\u003c/strong\u003e Calibration plot obtained from chronoamperomogram of glucose recorded for MWCNT-Fc-GOx/GCE.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/3d097d6cab626059a1dd3a2b.png"},{"id":57417309,"identity":"86b9a790-5eec-4de5-81d4-95b4a2f92193","added_by":"auto","created_at":"2024-05-30 11:57:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003ePower and current density curves at various concentrations with designed EBFC with the anode (MWCNT-Fc-GOx/GCE) and the cathode (Pt-Pd-Nps/KGE), \u003cstrong\u003eB)\u003c/strong\u003e Calibration plot obtained from power density values.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/6e3c2b6b5db8a1b408305087.png"},{"id":65628490,"identity":"b14cdaa3-a721-4346-bc68-c2d108e291d8","added_by":"auto","created_at":"2024-09-30 16:19:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1674348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/f3a616b2-4f5a-4497-8d8e-3e36e85f4e3e.pdf"},{"id":57417311,"identity":"792b84f6-11f1-4b38-bfb1-27eed1b71ef0","added_by":"auto","created_at":"2024-05-30 11:57:14","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1429167,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-4376605/v1/44d1b60e9abf937c9f224a4e.docx"}],"financialInterests":"","formattedTitle":"A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBiofuel cells (BFCs) are electrochemical devices that generate electrical energy using organic molecules produced during metabolic processes as a fuel and a renewable biological catalyst. They are considered environmentally friendly and sustainable energy options [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Two main BFCs have become prominent: enzymatic biofuel cells (EBFCs) that use purified oxidoreductase enzymes and microbial biofuel cells (MBFCs) that use microorganisms [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. EBFCs are attractive devices that directly convert the chemical energy stored in the fuel into electricity using enzymes as biocatalysts. These types of fuel cells are commonly employed in biosensors and bioelectronic devices. EBFCs transform enzymatic chemical reactivity into electrical power output by oxidizing fuel at the anode and reducing an oxidant at the cathode [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. EFBCs are known as renewable and environmentally friendly technologies due to their remarkable properties such as easy miniaturization, portability, working conditions around room temperatures and physiological pH. Furthermore, EBFCs offer advantages such as high conversion efficiency and versatility as they can generate electrical energy from various organic substrates [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Separating the anodic and cathodic parts is unnecessary thanks to the highly selective enzymes. This enables the development of miniature and membrane-less EBFCs implanted in the human body to potentially power medical devices [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, current approaches face several challenges, such as the need for specific pH and temperature conditions, difficulties in stabilizing enzymes to the electrode surfaces and inadequacies in electron transfer rate between the electrodes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous techniques have been developed to improve the enzyme immobilization, power output and stability of EBFCs. Various enzyme immobilization techniques, including direct immobilization, cross-linking with bifunctional linker molecules and self-assembled monolayers, have already been applied to EBFCs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Various electrode surface modifications were performed using novel materials to promote electron transfer between the redox enzyme and electrode [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For this reason, enzyme electrodes have been prepared using a variety of materials, like gold nanoparticles [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], carbon nanotubes, redox polymers, metal complexes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], or electron transfer mediators, which include osmium (Os), benzoquinone, polyvinyl ferrocene, ferrocene (Fc) and its derivatives [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Redox mediators are widely used in biosensors, energy conversion, bio-electrocatalysis and storage technologies. Ferrocene (Fc) and its derivatives are notable among all the mediators used due to their nontoxicity to the human body and solubility in various solvents, including water and ethanol [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Carbon nanotubes (CNTs) have also been used to improve the properties of Fc-modified electrodes because of their unique biocompatibility properties and excellent electrical conductivity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImproving the oxygen reduction reaction (ORR) activity is essential for fuel cell development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The glucose/O\u003csub\u003e2\u003c/sub\u003e biofuel cell, with a glucose oxidizing anode and an O\u003csub\u003e2\u003c/sub\u003e-reducing cathode, could become an \u003cem\u003ein vivo\u003c/em\u003e electricity source to power integrated medical devices. Recent developments in nanotechnology have led to the designing of new artificial enzymes called nanozymes. Nanozymes have numerous advantages, including diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, ease of surface tunability and biocompatibility. These advantages have allowed their integration into a wide range of biosensing applications [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Several biosensing systems have been developed using metal, metal oxide and metal-organic framework-based nanozymes [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this work, we designed a simple EBFC configuration operating in ambient and physiological conditions containing enzymatic composite material-based anode and nonenzymatic bimetallic nanoparticle-based cathode. Glucose was selected as the substrate due to its suitability as a biological fuel and its prevalence in various biological substrates such as blood and tears. In our system, glucose is oxidized by glucose oxidase (GOx) at the anode and oxygen is reduced by bimetallic nanoparticle composite material at the cathode. The anode consists of a GOx immobilized on a glassy carbon electrode (GCE) modified with an MWCNT and Fc. On the other hand, the cathode consisted of a pencil graphite electrode modified with platinum (Pt) and palladium (Pd) metal nanoparticles (MNps). Pencil graphite electrode (PGE) was preferred as an electrode material at the cathode because of its advantages, such as electrochemical activity, commercial accessibility, good mechanical strength, disposability, low cost, and simple modification procedure [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This novel design of EBFC has produced significant electrical power that can be used for various applications, including glucose detection or powering medical devices.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents\u003c/h2\u003e \u003cp\u003eAnalytical-grade chemicals were used without further purification. GOx (from \u003cem\u003eAspergillus niger\u003c/em\u003e type 77 units/mg) was purchased from Sigma Aldrich. Stock solutions of GOx were prepared using 0.1 M phosphate buffer solution (PBS) with pH 7.0, and stored at -20 \u003csup\u003e◦\u003c/sup\u003eC until used. Multi-walled carbon nanotubes, ferrocene, palladium (II) chloride (PdCl\u003csub\u003e2\u003c/sub\u003e), and chloroplatinic acid (H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e) solution 8% wt. in H\u003csub\u003e2\u003c/sub\u003eO was supplied from Sigma Aldrich.\u003c/p\u003e \u003cp\u003eChemicals such as potassium phosphate dibasic (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO), potassium phosphate monobasic (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO), potassium chloride (KCl), sodium hydroxide (NaOH), chitosan (medium molecular weight), D-(+)-glucose (\u0026ge;\u0026thinsp;99.5%) were purchased from Merck. Potassium ferricyanide (K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e].3H\u003csub\u003e2\u003c/sub\u003eO) and potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e].3H\u003csub\u003e2\u003c/sub\u003eO) were purchased from Sigma Aldrich. All solutions were prepared with ultrapure water using the Elga Option Q7B water purification system (18.2 \u0026micro;W cm\u003csup\u003e-1\u003c/sup\u003e). 0.10 M KCl containing 0.1 M PBS with pH 7.0 was used as an electrolyte to characterize electrodes and biofuel cell tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Apparatus and Electrochemical Measurements\u003c/h2\u003e \u003cp\u003eAll electrochemical measurements, including electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), linear sweep voltammetry (LSV), amperometry and open circuit potential (OCP) tests were run using a Metrohm model Autolab PGSTAT 128N Potentiostat/Galvanostat. Before carrying out the electrochemical tests and between each successive addition of glucose, it was ensured that the solutions were purged with either air or argon. The stock glucose solutions were allowed to mutarotate for at least 24 hours before use and then stored in a refrigerator at 4\u0026deg;C. CV and amperometric techniques individually characterized the electrodes employed as anodes and cathodes. In these cases, a three-electrode cell constituted by each unmodified or modified electrode as a working electrode, an Ag/AgCl (KCl, 3 M) as a reference electrode, and a platinum wire as an auxiliary electrode was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Fabrication of the Electrodes\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Preparation of Anode Electrodes (MWCNT-Fc-GOx/GCE)\u003c/h2\u003e \u003cp\u003eBefore a modification step, GCEs were polished with different grain sizes (1 \u0026micro;m, 0.3 \u0026micro;m, and 0.05 \u0026micro;m, respectively) alumina on a polishing cloth. Then, GCE was sonicated in ethanol and ultrapure water and dried with an IR lamp. Then, the dried electrodes were placed in an electrochemical cell, and a preconditioning step of 20 scans at 50 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between \u0026minus;\u0026thinsp;0.4 V and 0.4 V (vs. Ag/Ag\u003csup\u003e+\u003c/sup\u003e) was applied in 0.1 M KCl with the CV method. After the preconditioning step, the electrodes were washed with de-ionized water and dried in front of IR lamb for 5 minutes. After the cleaning procedure of GCE, 25 mM Fc containing 20 mg/mL MWCNT solution, was prepared and sonicated for 15 minutes in an ultrasonic bath. Then, 10.0 \u0026micro;L from that solution was added onto the GCE surface and dried with IR lamb for 5 minutes in every 1.0 mL addition. 20 mg/mL GOx mixed in 0.10 M PBS with pH 7.0, was then dropped on the surface of MWCNT-Fc-GCE, and dried for 2 hours at the room temperature. The preparation of the enzyme electrode is finalized by the addition of the % 0.5 chitosan solution in acetic acid. The enzyme electrodes were stored at +\u0026thinsp;4\u0026deg;C when not in use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Preparation of cathode electrodes (PtNps/PGE, PdNps/PGE and Pt-PdNps/PGE)\u003c/h2\u003e \u003cp\u003ePlatinum nanoparticles (PtNps) have been efficiently deposited onto the surface of PGE using an electrochemical method. It was achieved by recording cyclic voltammograms in a 5.0 mM solution of H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e containing 0.10 M KCl from 0.8 V to -0.8 V at a scan rate of 50 mVs\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 30 cycles. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). electrodeposition mechanism of PtCl\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e was reported by our previous study [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Palladium nanoparticles (PdNps) were also efficiently deposited onto the surface of PGE using an electrochemical method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cyclic voltammograms were recorded in a supporting electrolyte of 0.10 M KCl and 5.0 mM PdCl\u003csub\u003e2\u003c/sub\u003e at a scan rate of 25 mVs\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 30 cycles within a potential range of 0.40 V to -0.25 V (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). For the PtPd-Nps electrodes, 5.0 mM of H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e and 5.0 mM of PdCl\u003csub\u003e2\u003c/sub\u003e containing 0.1 M KCl solutions were used. This is also achieved by recording CV under the same conditions as PtNps and PdNps deposition, respectively (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization of Electrodes\u003c/h2\u003e \u003cp\u003eIn this study, SEM and EDX images of both unmodified and modified PGE electrodes were taken to provide an accurate analysis of the surface changes resulting from the electrochemical processes. Scanning electron microscopy (SEM-EDX) images was taken at Experimental Science Application and Research Center (DEFAM) of Manisa Celal Bayar University with a device of Zeiss, Gemini 500 model. Also, the electrodes were electrochemically characterized using CV and EIS techniques, which were recorded in a solution containing 5.0 mM Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e + 0.10 M KCl.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Fuel Cell Tests and Real Sample Analysis\u003c/h2\u003e \u003cp\u003eThe fully assembled biofuel cells were characterized by recording LSV using MWCNT-Fc-GOx/GCE as anode and Pt-PdNps/PGE as a cathode in oxygen-saturated 0.1 M PBS at pH 7.0 with different glucose concentrations. LSV was performed at 1.0 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from the OCP of the cell to 0 V to evaluate the performance of the designed EBFCs. The current and power of the fuel cell were calculated using Ohm's Law (V\u0026thinsp;=\u0026thinsp;I \u0026times; R and P\u0026thinsp;=\u0026thinsp;I \u0026times; V). The output density and current density were calculated from the apparent surface area of the anode. All the measurements were carried out at 37 \u0026ordm;C.\u003c/p\u003e \u003cp\u003eThe constructed glucose-based self-powered biofuel cell was tested using a sample of artificial blood serum containing 4.77 mM glucose. The performance of the designed EBFCs in real samples was evaluated using LSV.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of Nanozyme-Based Cathode (Pt-Pd-Nps/PGE) and Its Electrochemical Response to ORR\u003c/h2\u003e \u003cp\u003eIn the first stage of the studies, the characterization of the cathode was carried out, and its electrocatalytic activity with the reduction of O\u003csub\u003e2\u003c/sub\u003e was examined. It is well known that biocathodes consisting of enzymes such as bilirubin oxidase and laccase have been extensively used in developing EFCs due to their direct electron transfer ability and selectivity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These properties allow them to operate without a separation membrane to prevent cross-reactions at the electrodes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Enzyme-mimicking nanomaterials called nanozymes have recently been introduced to biosensor and fuel cell studies [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, PGEs modified with Pt-Pd bimetallic nanoparticles used as nanozyme have been proposed as a new cathode material in biofuel cell studies for the first time.\u003c/p\u003e \u003cp\u003eFirst, PtNps, PdNps and Pt-PdNps were modified on PGE using electrochemical methods. SEM images and EDX spectra were then recorded for unmodified and modified PGEs. SEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) show that the layered structures of unmodified PGE are homogeneously covered with nanoparticles obtained from monometal and bimetal deposition. According to EDX results (Fig. S2), it is expected to see %76.93 C, %17.68 O, %0.68 Mg, %1.53 Al, % 0.25 Ca, %2 Fe and %2.75 Si in unmodified PGE because pencil leads consist of clay and graphite [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, 13.38% Pt in PtNps, 8.52% Pd in PdNps, and 4.27% Pt and 16.47% Pd in Pt-PdNps were found, which reflects that monometallic and bimetallic nanoparticles were successfully attached to the PGE surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEIS is widely used to understand electrode surface changes and resistance properties. The EIS curve consists of two main parts: the semicircle and the linear portion. These parts provide detailed information on the impedance changes of an electrode surface, which is valuable for understanding modifications of electrode surfaces and their use in electrochemical sensing applications [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The linear portion of impedance spectra corresponds to the processes of electron transfer that are limited by diffusion. The semicircle is closely related to high frequencies, and its diameter provides information about the charge transfer resistance (R\u003csub\u003ect\u003c/sub\u003e), which controls the electron transfer kinetics of the redox probe at the electrode's surface [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, unmodified and modified PGEs were also electrochemically characterized by evaluating their CVs and EIS to understand electrode surface changes and surface resistance properties. EIS spectra of unmodified Pt-Nps/PGE, Pd-Nps/PGE, and PtPd-NPs/PGE were recorded in 10.0 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3-/4-\u003c/sup\u003e containing 0.10 M KCl. The R\u003csub\u003ect\u003c/sub\u003e values obtained from Nyquist curves were obtained as 110, 37, 18 and 8 ohms for unmodified PGE, Pd-Nps/PGE, Pt-Nps/PGE, and PtPd-NPs/PGE, respectively (Fig. S3A). This is attributed to the fact that the metal nanoparticles speed up the transfer of electrons on the surface of the PGE. CVs recorded in the redox probe for each electrode agree well with the EIS measurements. It can be seen from CVs that the highest peak currents of the redox probe were obtained from Pt-PdNps/PGE, while the lowest peak currents were obtained from unmodified PGE (Fig. S3B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo compare the electrocatalytic activities of the electrodes (bimetallic NPs-modified PGE), ORR was performed by using unmodified PGE and monometallic modified PGEs (PtNps/PGE and PdNps/PGE) together with Pt-PdNps/PGE as cathodes. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the CVs and LSVs of these electrodes in the argon and oxygen-saturated 0.1 M KCl containing 0.1 M PBS at pH 7.0. It can be clearly seen that a remarkable enhancement in the electrocatalytic reduction of O\u003csub\u003e2\u003c/sub\u003e was observed at Pd-PtNps/PGE compared to unmodified PGE and the monometallic NPs-modified electrode. On the other hand, ORR has a high overpotential at unmodified PGE, and the current starting at 0 mV did not take the form of a peak but gradually increased by shifting potential in a more negative direction. ORR current of PtNps/PGE, PdNps/PGE, and Pt-PdNps/PGE were found to be 280, 340, and 570 \u0026micro;A at -50, +\u0026thinsp;50, and +\u0026thinsp;90 mV vs. Ag/AgCl, respectively. It was concluded that Pt-PdNps/PGE is the most suitable electrode for the cathode in glucose/O\u003csub\u003e2\u003c/sub\u003e type EBFC because bimetallic nanoparticle-modified PGE has the highest peak current at a more positive voltage in the reduction potential compared to monometallic-modified PGEs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization and Electrochemical Behavior of Bioanode (MWCNT-Fc-GOx/GCE)\u003c/h2\u003e \u003cp\u003eDirect electron transfer becomes difficult for GOx since the electroactive group is in the inner parts of the enzyme [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To overcome this situation, electrodes are modified with redox mediators such as ferrocene derivatives and osmium redox polymers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Redox mediators facilitate electron transfer rates between substrate and electrode, resulting in an enhancement in electron transfer at the bioanaode surface and in output power. In this study, ferrocene was immobilized with GOx onto MWCNT/GCE to obtain an effective bioanode.\u003c/p\u003e \u003cp\u003eTo obtain the optimum MWCNT-Fc-GOx/GCE, which gives the most effective response to glucose oxidation, the GCE surface was optimized by adjusting the different concentrations of MWCNT, Fc and GOx during the electrode preparation step. To determine the optimum electrode composition, CVs of each electrode prepared with different ratios of modification reagents were recorded in the absence and presence of 5.0 mM glucose. Fig. S4. shows the curves of the concentration of modification reagents versus the electrocatalytic oxidation peak current of glucose. The maximum peak current for the electrocatalytic oxidation of glucose was achieved when MWCNT, Fc, and GOx (concentrations of 5.0 mg/mL, 25.0 mM, and 20.0 mg/mL, respectively) were immobilized at GCE surface.\u003c/p\u003e \u003cp\u003eThe obtained optimum MWCNT-Fc-GOx/GCE was characterized by recording EIS and CVs in the 5.0 mM Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e4-/3-\u003c/sup\u003e redox probe containing 0.10 M KCl. The Nyquist curves were used to estimate the R\u003csub\u003ect\u003c/sub\u003e, which was then used to evaluate the insulating properties of the electrode/electrolyte interface. Fig. S5A shows that the R\u003csub\u003ect\u003c/sub\u003e values were found to be 1610 (Fig. S5A/c), 140 (Fig. S5A/b), and 580 (Fig. S5A/c) ohms for unmodified GCE, MWCNT-Fc-GCE and MWCNT-Fc-GOx-GCE, respectively. R\u003csub\u003ect\u003c/sub\u003e value of unmodified GCE (1610 ohm) was remarkably decreased after the modification of both MWCNT and Fc (140 ohm) due to high surface area and conductivity of MWCNT and electrostatic interaction between positively charged Fc and negatively charged redox probe. These results indicate that the conductivity of the MWCNT-Fc/GCE, which is directly related to its electron transfer ability, is higher than that of the unmodified GCE. When the surface of MWCNT-Fc/GCE is immobilized with GOx, R\u003csub\u003ect\u003c/sub\u003e is increased to 580 ohms due to steric hindrance between the redox probe and large GOx. In addition, there is good agreement between the CV results given in Fig. S5B and the EIS measurements. CVs from Fig S5B show that the highest peak currents of the redox probe were obtained from MWCNT-Fc/GCE, while the lowest peak currents were obtained from MWCNT-Fc-GOx/GCE. The information obtained from the voltammograms and impedance spectra indicates that MWCNT and Fc play an important role in increasing the electron transfer and conductivity properties between GCE and the Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3-/4-\u003c/sup\u003e redox probe. Finally, the modification procedure with MWCNT-Fc and GOx was successful.\u003c/p\u003e \u003cp\u003eGOx is often used as a biocatalyst to produce biosensors and EBFCs. To evaluate the efficacy of MWCNT-Fc-GOx/GCE in catalyzing glucose oxidation, a series of comparative CVs were recorded in the absence and presence of glucose in a 0.1 M PBS with pH 7.0 containing 0.1 M KCl at a scan rate of 20 mVs\u003csup\u003e-1\u003c/sup\u003e. It is expected that CV recorded by an unmodified GCE showed no anodic peaks attributed to glucose oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB/a shows that the response of MWCNT-Fc-GOx/GCE in the absence of glucose. A well-defined redox couple with a formal potential of 300 mV indicates high activity and shows a characteristic, reversible redox peak behavior of Fc, in which a single electron transfer is involved for the electro-oxidation peak current of Fc. Also, electrocatalytic oxidation of glucose was observed at the GOx immobilized on the MWCNT-Fc/GCE surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB/b). The anodic peak of the MWCNT-Fc-GOx/GCE remarkably increased in the presence of 10 mM of glucose, while in the presence of the same glucose concentration, the cathodic peaks slightly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB/b). Therefore, the significant increase in the anodic peak of the modified electrodes when exposed to glucose demonstrates the excellent oxidative electrocatalytic activity of the GOx with electrocatalyst (Fc) towards glucose. Considering that the GCE area, which is 0.07 cm\u0026sup2;, results in a high current density, fast charge transfer on the GCE proves its suitability for electrode modification with GOx, Fc, and MWCNT. CVs of MWCNT-Fc-GOx/GCE were also measured at different glucose concentrations ranging from 0 to 20 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The electrodes exhibited a higher oxidative current response with increasing glucose concentrations. They showed the same onset potential (approximately \u0026minus;\u0026thinsp;0.1 V vs. Ag/Ag\u003csup\u003e+\u003c/sup\u003e) in the CV test, which is dependent on the activation of the glucose oxidation reaction. The successful immobilization of enzymes on the electrode surface and electron transfer between the enzyme and electrode via Fc mediator is suggested to have been achieved for Fc-MWCNTs modified electrodes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe electrocatalytic glucose oxidation at the enzymatic electrode (MWCNT-Fc-GOx/GCE) was also investigated by chronoamperometry (CA). To assess the effect of applied potential on the response to glucose, CAs were recorded for MWCNT-Fc-GOx/GCE at various potentials ranging from 0.20 to 0.5 V. This was achieved by adding 1 mM glucose successively into a continuously stirred 10 mL solution of 0.10 M pH 7.0 PBS containing 0.1 M KCl. Results show that maximum peak current was obtained when the applied potential was fixed 0.3 V vs Ag/AgCl. CAs of MWCNT-GOx/GCE and MWCNT-Fc-GOx/GCE at 0.30 V were recorded based on the successive addition of glucose in the range from 1 to 10 mM. The CAs in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA indicate a steady-state current was recorded within approximately 5 seconds of adding glucose. Additionally, it can be observed that these steady-state currents increased as the glucose concentration increased. The MWCNT-GOx/GCE (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, red line) does not respond to glucose. However, the MWCNT-Fc-GOx/GCE electrode responds well to glucose. The signal recorded by the modified GCE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, black line) was higher than that of the MWCNT-GOx/GCE. The calibration plot suggests a linear relationship between current and glucose concentration, which varied from 1 to 10 mM during the study of MWCNT-Fc-GOx/GCE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To calculate the limit of detection (LOD), limit of quantification (LOQ) and sensitivity values of the proposed sensor, the lowest concentration that gives a amperometric response was recorded ten times. The LOD and LOQ were calculated according to the equation of 3xSD/m and 10xSD/m,(\u0026ldquo;SD\u0026rdquo;: the standard deviation for peak currents and \u0026ldquo;m\u0026rdquo;: the slope calculated from the calibration curve) and determined as 0.261 mM and 0.87 mM, respectively. Also sensitivity of the proposed sensor were calculated 25.71 \u0026micro;A/mM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the final step of anode characterization, the influence of potentially interfering compounds, such as other monosaccharides (galactose, mannose, fructose, maltose), disaccharides (sucrose), uric acid (UA), dopamine (DA), and ascorbic acid (AA), on the response to glucose was examined at MWCNT-Fc-GOx/GCE. For this purpose, the steady-state background current was stabilized under the optimized conditions. The CAs of MWCNT-Fc-GOx/GCE were recorded by adding known volumes of stock solutions of glucose and interfering compounds to an electrochemical cell. The concentrations of glucose and each interfering compound were fixed at the same concentration. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e demonstrates the response of interference molecules toward glucose. The results show that the designed anode electrode was minimally affected by fructose, sucrose, maltose, mannose, galactose, glutamic acid, lactose, and sodium lactate while highly affected by UA, AA, and DA. It is well known that these substances exhibit positive interference effects by oxidizing at high anodic potentials and enhancing the glucose biosensor's signal. Our previous studies showed that a pre-oxidant (NaBiO\u003csub\u003e3\u003c/sub\u003e) was successfully used to minimize the interference of these substances [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, solutions, including glucose and interference substances, were passed from NaBiO\u003csub\u003e3\u003c/sub\u003e filled in an injector several times. After that, the interference effects of UA, DA, and AA were significantly reduced by their pre-oxidation with NaBiO\u003csub\u003e3\u003c/sub\u003e, while glucose was not oxidized by NaBiO\u003csub\u003e3\u003c/sub\u003e. The results indicate that MWCNT-Fc-GOx/GCE demonstrates a high selectivity towards glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Fuel Cell Performance and Real Sample Analysis\u003c/h2\u003e \u003cp\u003eGlucose was chosen as a biofuel model in our BFC development because it is one of the significant components of human biofluids, including sweat. The fuel cell performance of the system was evaluated by monitoring power output and current density dependent on OCP in air-saturated PBS containing 0.10 M KCl and various concentrations of glucose in the range from 1 to 10 mM. The maximum current density of 460 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e and a maximum power output of 32.05 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e were obtained at a voltage of 0.285 V (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The high current and power density were achieved due to the customized composite electrodes in both the anode and cathode. These electrodes possess desirable electrical conductivity and electrocatalytic properties. The hybrid MWCNT-Fc enables an efficient transfer of electrons from the anode to the cathode, significantly increasing the power output. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB demonstrates a positive correlation between glucose concentration and power density within the 0\u0026ndash;10 mM glucose range. The response generated by the BFC was observed with successive additions of glucose in the range of 0\u0026ndash;10 mM. The calibration plot for the self-generated current signal demonstrates linearity up to 10 mM glucose. This observation provides strong evidence for the ability of the BFC to detect glucose levels in this range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. compares the performance of the designed \u003cb\u003eglucose-based\u003c/b\u003e EBFC with the previously reported EBFCs. As can be seen from Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the proposed \u003cb\u003eglucose-based\u003c/b\u003e EBFC demonstrates better performance than some other previously reported EBFCs in terms of power density values. Furthermore, the designed electrode for the cathode has never been used \u003cb\u003epreviously\u003c/b\u003e for ORR. PGEs have \u003cb\u003eseveral\u003c/b\u003e benefits, including disposability, affordability, mechanical rigidity, simplicity, commercial availability, and ease of surface modification [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cb\u003eAlso, nanozymes have many advantages such as d\u003c/b\u003eiverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, ease of surface tunability and biocompatibility. \u003cb\u003eTherefore, our cathode design brings the advantages of PGE and nanozymes together.\u003c/b\u003e The mechanism of Pt-Pd-NpsPGE boosting the electrochemical reduction of oxygen can be attributed to the synergistic effect of Pt and Pd nanoparticles, which makes oxygen reduction at the cathode surface easier. Additionally, the cathode electrode can reduce oxygen to water without the need for enzymes such as BOD and Lc. The investigation was conducted to determine the performance of the designed EBFC with artificial serum samples. To attain the desired outcome, the glucose levels in artificial serum samples were measured by the LSV method, and the voltammograms for each sample were recorded under optimized conditions. At 0.280 V, a maximum current density of 290 \u0026micro;A/cm\u003csup\u003e2\u003c/sup\u003e and a maximum power output of 11.35 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e was achieved using 10 mM glucose (Fig. S6). This study has shown that designed EBFC can be effectively used for glucose determination in real and artificial samples and suggests their potential as simple to configure energy-sustaining and non-invasive glucose monitoring devices.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the analytical performance of proposed self-powered EBFC with some previously reported EBFCs in the literature.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBFC/analyte\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCathode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOCP (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePower Density\u003c/p\u003e \u003cp\u003e(mW/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCurrent Density (mA/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGDH/poly(TBO)/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGOx/HRP/MWCNT/GE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e650.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e165.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGRE/5AP/GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGRE/HRP/GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e450.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePGE/MWCNT/GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePGE/MWCNT/laccase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e433.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePPD/(AuNPs)PPCA‒GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGRE/PB-PPCA/PPCA‒GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e640.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGDH-MB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGOx-hemin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e230.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePPy\u0026ndash;GOD\u0026ndash;CNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePPy\u0026ndash;CNT\u0026ndash;laccase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMWCNT-PCA/Ca\u003csup\u003e2+\u003c/sup\u003e/GOx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003erGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e140.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose/O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFc-Go/GC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBOD-ABTS\u003csup\u003e2\u0026minus;\u003c/sup\u003e/fBP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlucose/O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMWCNT-Fc-GOx/GCE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePtPd-Nps/PGE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e285.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e32.05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e460.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eIn this work\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e(rGC: Reduced graphene oxide-ceria, MWCNT-PCA: Multiwalled carbon nanotube-pyrene carboxylic acid, Fc: Ferrocene, GC: Glassy Carbon Electrode, BOD: Bilirubin Oxidase, ABTS: 2,2\u0026prime;-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt, fBP: functionalized bucky paper, Pt-Pd-Nps: Pt-Pd Nanoparticles, PPy: Poly-Pyrrole, Box: Bilirubin Oxidase, GDH: Glucose Dehydrogenase, MB: Methylene blue, PPD: poly(1,10-phenanthroline-5,6-dione), PB: Prussian blue, PPCA: poly(pyrrole-2-carboxylic acid), MWCNT: Multi-walled carbon nanotube, HRP: Horseradish peroxidase)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eCombining bimetallic and enzymatic catalysts has been suggested as a unique approach to moderate-condition glucose oxidation. This study presents the first evaluation of PGE modified with Pt-Pd bimetallic nanoparticles for oxygen reduction at the cathode. Furthermore, the membraneless design of our EBFC presents another advantage for the developed system. When glucose concentration is 10 mM or higher, the EBFC exceeds a maximal registered open-circuit voltage of 0.285 V. The maximal power density was calculated to be 32.05 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e when the cell voltage was 0.285 V and [glucose] was 10 mM. This could be attributed to the enhanced energetic efficiency due to the use of Fc serving as a mediator for GOx and Pt-Pd bimetallic Nps for ORR. Also, MWCNT can significantly enhance the efficacy of EBFCs by increasing the effective electrode area. Therefore, the EBFC system proposed here may open the possibility of developing more effective energy conversion and applicability to real samples.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthical Approval:\u003c/em\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent to Participate:\u003c/em\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for Publication\u003c/em\u003e: We declare that the information in this manuscript has not been published elsewhere nor is it under consideration by any other journal. Furthermore, it is the consensus of all authors to submit this manuscript for possible publication in ABB.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor Contributions:\u003c/em\u003e Gamze Emir: Investigation, methodology, analysis and writing original draft; Samet Şahin: Editing, review and supervision; Yusuf Dilgin: Editing, review and supervision; Cahit Akg\u0026uuml;l: Writing, review \u0026amp; editing, conceptualization, supervision.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding:\u0026nbsp;\u003c/em\u003eWe appreciate the Scientific Research Projects Coordination Unit of \u0026Ccedil;anakkale Onsekiz Mart University (FDK-2022-4073) for financial support. This study was produced as a part of the PhD thesis of Gamze Emir GUNAY.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting Interests:\u003c/em\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e: The data that support the findings of this study are not openly available and are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarelli, L., Bidini, G., Pelosi, D., \u0026amp; Sisani, E. (2021). Energies, 14(4), 910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChengcheng, G., Gai, P., \u0026amp; Li, F. (2022). \u003cem\u003eNano Energy\u003c/em\u003e, 93, 106806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahin, S., Wongnate, S. T., Chuaboon, L., Chaiyen, P., \u0026amp; Yu, E. H. 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(2014). \u003cem\u003eJournal Of Power Sources\u003c/em\u003e, 247, 579\u0026ndash;586.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Enzymatic biofuel cell (EBFC), Glucose Oxidase (GOx), Nanozyme, Self-Powered Glucose Sensor (SPGS), Ferrocene (Fc), Bimetallic Nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-4376605/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4376605/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnzymatic biofuel cells (EBFC) are promising sources of green energy owing to the benefits of using renewable biofuels, eco-friendly biocatalysts, and moderate operating conditions. In this study, a simple and effective EBFC was presented using an enzymatic composite material-based anode and a nonenzymatic bimetallic nanoparticle-based cathode, respectively. The anode was constructed from a glassy carbon electrode (GCE) modified with a multi-walled carbon nanotube (MWCNT) and ferrocene (Fc) as a conductive layer coupled with the enzyme glucose oxidase (GOx) as a sensitive detection layer for glucose. A chitosan layer was also applied to the electrode as a protective layer to complete the composite anode. On the other hand, the cathode consisted of a disposable pencil graphite electrode (PGE) modified with platinum-palladium bimetallic nanoparticles (Nps) which exhibit excellent conductivity and electron transfer rate for the oxygen reduction reaction. The EBFC consisting of MWCNT-Fc-GOx/GCE anode and Pt-PdNps/PGE cathode exhibits an open circuit potential of 285 mV and a maximum power density of 32.25 \u0026micro;W cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e under optimized conditions. The results show that the proposed EBFC is a promising candidate for detecting glucose while harvesting power from artificial serum samples.\u003c/p\u003e","manuscriptTitle":"A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 11:57:09","doi":"10.21203/rs.3.rs-4376605/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-19T04:43:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-19T22:28:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2024-05-08T08:37:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2024-05-07T08:39:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eb45c63e-d202-40d2-b025-157d22e71d80","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:15:05+00:00","versionOfRecord":{"articleIdentity":"rs-4376605","link":"https://doi.org/10.1007/s12010-024-05068-1","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2024-09-27 15:58:00","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-05-30 11:57:09","video":"","vorDoi":"10.1007/s12010-024-05068-1","vorDoiUrl":"https://doi.org/10.1007/s12010-024-05068-1","workflowStages":[]},"version":"v1","identity":"rs-4376605","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4376605","identity":"rs-4376605","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}