Construction and Application of a Non-Enzyme Hydrogen Peroxide Electrochemical Sensor Based on Eucalyptus Porous Carbon

Natural eucalyptus biomorphic porous carbon (EPC) materials with unidirectional ordered pores have been successfully prepared by carbonization in an inert atmosphere. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) were employed to characterize the phase identification, microstructure and morphology analysis. The carbon materials were used to fabricate electrochemical sensors to detect hydrogen peroxide (H2O2) without any assistance of enzymes because of their satisfying electrocatalytic properties. It was immobilized on a glassy carbon electrode (GCE) with chitosan (CHIT) to fabricate a new kind of electrochemical sensor, EPC/CHIT/GCE, which showed excellent electrocatalytic activity in the reduction of H2O2. Meanwhile, EPC could also promote electron transfer with the help of hydroquinone. The simple and low-cost electrochemical sensor exhibited high sensitivity, and good operational and long-term stability.


Introduction
The accurate and sensitive detection of H 2 O 2 content is of great significance in clinical diagnostics, food safety, environmental monitoring and other fields [1]. Therefore, more detection techniques such as titration analysis [2], spectrophotometry [3], chemiluminescence [4], and electrochemical methods [5] are used to analyze H 2 O 2 . Among these methods, electrochemical methods are widely used and are widely reported. It is generally believed that ampere-based electrochemical biosensors based on fixed horseradish peroxidase (HRP) are the most effective and commonly used methods for detecting H 2 O 2 . However, the denaturation and shedding of surface enzymes on the sensor electrode of immobilized enzymes and other bioprotein molecules has been a major obstacle to the application of enzyme sensors, especially commercial applications, because it directly affects the sensitivity, stability and repeatability of the sensor. It is still a key issue in enzyme sensors that has to be overcome so far. At present, the immobilization of an inorganic material with a direct electrocatalytic activity and a very stable nature to the surface of the electrode to construct a non-enzymatic sensor can effectively overcome the problems caused by the enzyme-based sensor, since the material with stable properties does not undergo any denaturation. Some non-enzymatic sensors for detecting H 2 O 2 have been reported in the literature. For example, metals like silver platinum, gold or their combination [6][7][8][9][10][11]; WS 2 [12] and electrogenerated ferrites or their combination with carbon nanomaterials [13,14] have been successfully used for the analytical determination of hydrogen peroxide.
The original ecological eucalyptus was a carbonized material for the preparation of biomorphic porous carbon. Chitosan (CHIT, MW~1 × 10 6 , >85% deacetylated) and hydroquinone were purchased from Sigma (Shanghai, China). Hydrogen peroxide (H 2 O 2 , 30% V/V aqueous solution) was obtained from Shanghai Biochemical Reagent Company (Shanghai, China). Other analytical grade chemicals were purchased from Beijing Chemical Reagent Company (Beijing, China) and did not require further purification. Phosphate buffer solution (PBS, 0.067 M) was prepared by using K 2 HPO 4 and NaH 2 PO 4 standard solutions and adjusting the pH to 7.0. All solutions were prepared with deionized water.
The electrochemical experiment was carried out on a CHI660B electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., Shanghai, China) under normal temperature conditions, and a three-electrode system was used: A platinum electrode as a counter electrode; a KCl-saturated Ag/AgCl electrode as a reference electrode; and a 3 mm diameter glassy carbon electrode as the working electrode. A scanning electron microscope (SEM) image was obtained by JEM-6700F (JEOL, Tokyo, Japan). Fourier transform infrared spectroscopy (FT-IR) was obtained by using KBr as a compression background on a WQF-410 infrared spectrometer (Beijing Second Optical Instrument Co., Ltd., BeiJing, China). The XRD of EPC was characterized by the Siemens D5000 (München, Germany).

Preparation of EPC Materials
The processed eucalyptus block with a diameter of 5-10 mm was first dried at 120 • C for 48 h and then heated to 600 • C at a heating rate of 2 • C/min and maintained at 600 • C 3 h to completely decompose the polyaromatic complex into carbon; The temperature was raised to 1200 • C at a temperature rise rate of 5 • C/min, and the temperature was maintained for 4 h to produce crack-free carbon; finally, the eucalyptus porous carbon (EPC) material was obtained by naturally cooling to room temperature. It is to be noted that the entire process was carried out under inert gas protection conditions.

Preparation of EPC/CHIT/GCE Electrochemical Sensors
Prior to electrode modification, the glassy carbon electrode was subjected to a series of grinding processes on alumina powders of different particle sizes, ultrasonically washed with ethanol and water, and finally blown dry with nitrogen. After the electrode was processed, the EPC was first ultrasonically dispersed with water to obtain a uniform dispersion with a concentration of 1 mg/mL. After thoroughly mixing the 0.5%-wt CHIT solution with the EPC suspension, 5 µL of the above mixed droplets were taken out and added to the surface of the glassy carbon electrode, and naturally dried to obtain EPC/CHIT/GCE. The sensor was placed in PBS solution and stored at room temperature.
As a control experiment, a bare glassy carbon electrode and a CHIT modified glassy carbon electrode (CHIT/GCE) were prepared by a method similar to that described above.

Characterization of EPC Materials
The eucalyptus was composed of dense cells with small pores, and the interstitial structure was uniform and dense. The carbonized EPC still maintained this regular and orderly structure. Figure 1 is a Scanning Electron Microscope (SEM) image of the EPC and EPC/CHIT/GCE. From Figure 1A we can see that the EPC has a honeycomb shape and is a hollow channel structure with different diameters. This structure is derived from the structure of the natural form of eucalyptus. The black part of the figure is the cell cavity of the eucalyptus, while the gray part is the cell wall of the eucalyptus. Most of the holes have an elliptical topography and are connected to each other through the wall of the hole. These cells are parallel to the axial direction of the elm texture structure ( Figure 1B). The anisotropic microstructure of EPC is different from other natural porous materials (such as zeolite, diatomaceous earth, etc.), and this dense one-way natural pore structure greatly increases its specific surface area. The structural morphology of the EPC/CHIT/GCE on the electrode surface was clearly seen by scanning electron microscopy ( Figure 1C). The material maintains its unique three-dimensional structure even on the electrode surface, which is porous. The array layout hardly changed. ultrasonically dispersed with water to obtain a uniform dispersion with a concentration of 1 mg/mL. After thoroughly mixing the 0.5%-wt CHIT solution with the EPC suspension, 5 μL of the above mixed droplets were taken out and added to the surface of the glassy carbon electrode, and naturally dried to obtain EPC/CHIT/GCE. The sensor was placed in PBS solution and stored at room temperature. As a control experiment, a bare glassy carbon electrode and a CHIT modified glassy carbon electrode (CHIT/GCE) were prepared by a method similar to that described above.

Characterization of EPC Materials
The eucalyptus was composed of dense cells with small pores, and the interstitial structure was uniform and dense. The carbonized EPC still maintained this regular and orderly structure. Figure 1 is a Scanning Electron Microscope (SEM) image of the EPC and EPC/CHIT/GCE. From Figure 1A we can see that the EPC has a honeycomb shape and is a hollow channel structure with different diameters. This structure is derived from the structure of the natural form of eucalyptus. The black part of the figure is the cell cavity of the eucalyptus, while the gray part is the cell wall of the eucalyptus. Most of the holes have an elliptical topography and are connected to each other through the wall of the hole. These cells are parallel to the axial direction of the elm texture structure ( Figure  1B). The anisotropic microstructure of EPC is different from other natural porous materials (such as zeolite, diatomaceous earth, etc.), and this dense one-way natural pore structure greatly increases its specific surface area. The structural morphology of the EPC/CHIT/GCE on the electrode surface was clearly seen by scanning electron microscopy ( Figure 1C). The material maintains its unique threedimensional structure even on the electrode surface, which is porous. The array layout hardly changed. As can be seen from the XRD of the EPC (Figure 2A), there are two broad peaks appearing at 24° and 44°, respectively assigned to the (002) peak (101) peak of amorphous carbon. These two characteristic peaks indicate that the material after carbonization is amorphous carbon [17]. It was found by FT-IR absorption spectroscopy ( Figure 2B) that the EPC contained more functional groups. The main absorption bands of the synthetic materials in the range of 400-4000 cm −1 were 3135, 1706, 1564 and 1175 cm −1 , which are attributed to the stretching vibration of hydroxyl groups, the stretching vibration of carbonyl groups, C-O (hydroxyl, ester, or ether) stretching and O-H bending vibration, respectively [18]. The N2 adsorption-desorption isotherms and X-ray photoelectron spectroscopy of the EPC materials are shown in Figure 2C,D. The nitrogen adsorption-desorption isotherms show an obvious hysteresis loop, which indicates that the EPC material is porous. The surface area of the EPC materials was 598.1 m 2 ·g −1 , calculated using the Brunauer-Emmett-Teller (BET) equation. The X-ray photoelectron spectroscopy (XPS) showed that the EPC material contained a small amount of iron in addition to carbon and oxygen ( Figure 2D). As can be seen from the XRD of the EPC (Figure 2A), there are two broad peaks appearing at 24 • and 44 • , respectively assigned to the (002) peak (101) peak of amorphous carbon. These two characteristic peaks indicate that the material after carbonization is amorphous carbon [17]. It was found by FT-IR absorption spectroscopy ( Figure 2B) that the EPC contained more functional groups. The main absorption bands of the synthetic materials in the range of 400-4000 cm −1 were 3135, 1706, 1564 and 1175 cm −1 , which are attributed to the stretching vibration of hydroxyl groups, the stretching vibration of carbonyl groups, C-O (hydroxyl, ester, or ether) stretching and O-H bending vibration, respectively [18]. The N 2 adsorption-desorption isotherms and X-ray photoelectron spectroscopy of the EPC materials are shown in Figure 2C,D. The nitrogen adsorption-desorption isotherms show an obvious hysteresis loop, which indicates that the EPC material is porous. The surface area of the EPC materials was 598.1 m 2 ·g −1 , calculated using the Brunauer-Emmett-Teller (BET) equation. The X-ray photoelectron spectroscopy (XPS) showed that the EPC material contained a small amount of iron in addition to carbon and oxygen ( Figure 2D).

Cyclic Voltammetry
The properties of EPC that promoted electron transfer were first investigated in PBS containing 1 mM hydroquinone. Figure 3 summarizes the electron transfer characteristics of different modified electrodes. Under the condition of −0.2 V operating potential with Ag/AgCl and no hydrogen peroxide, a pair of hydroquinone redox peaks appeared in different modified electrodes. It is apparent from the figure that the redox peak of CHIT/GCE (curve a, Figure 3) was smaller than the bare GCE (curve b, Figure 3), which indicates that CHIT itself may hinder electron transport. However, EPC/CHIT/GCE had a pair of redox peaks with significantly larger current values and a smaller peak difference (curve c, Figure 3). The electrocatalytic activity of EPC/CHIT/GCE for H2O2 was further investigated by cyclic voltammetry. In the absence of hydrogen peroxide, a typical redox characteristic peak of hydroquinone (curve a, Figure 4) occurred when a certain concentration of hydrogen peroxide was added to the PBS at about −0.2 V (vs. Ag/AgCl) showed a significant increase in the reduction peak current, accompanied by a decrease in the oxidation peak current, and as the H2O2 concentration in the system increased, the reduction peak current also increased (curve b to f, Figure 4). The experimental results show that EPC can be used as an excellent non-enzymatic catalyst for the construction of non-enzyme electrochemical sensors for detecting H2O2. It was fully demonstrated that EPC can promote the transfer of electrons between the catalytically active center and the electrode surface. The catalytically active center may be present in the three-dimensional biomorphological structure of the pore structure or the surface of the material.

Cyclic Voltammetry
The properties of EPC that promoted electron transfer were first investigated in PBS containing 1 mM hydroquinone. Figure 3 summarizes the electron transfer characteristics of different modified electrodes. Under the condition of −0.2 V operating potential with Ag/AgCl and no hydrogen peroxide, a pair of hydroquinone redox peaks appeared in different modified electrodes. It is apparent from the figure that the redox peak of CHIT/GCE (curve a, Figure 3) was smaller than the bare GCE (curve b, Figure 3), which indicates that CHIT itself may hinder electron transport. However, EPC/CHIT/GCE had a pair of redox peaks with significantly larger current values and a smaller peak difference (curve c, Figure 3). The electrocatalytic activity of EPC/CHIT/GCE for H 2 O 2 was further investigated by cyclic voltammetry. In the absence of hydrogen peroxide, a typical redox characteristic peak of hydroquinone (curve a, Figure 4) occurred when a certain concentration of hydrogen peroxide was added to the PBS at about −0.2 V (vs. Ag/AgCl) showed a significant increase in the reduction peak current, accompanied by a decrease in the oxidation peak current, and as the H 2 O 2 concentration in the system increased, the reduction peak current also increased (curve b to f, Figure 4). The experimental results show that EPC can be used as an excellent non-enzymatic catalyst for the construction of non-enzyme electrochemical sensors for detecting H 2 O 2 . It was fully demonstrated that EPC can promote the transfer of electrons between the catalytically active center and the electrode surface. The catalytically active center may be present in the three-dimensional biomorphological structure of the pore structure or the surface of the material.   Figure 5 shows the electrochemical response of EPC/CHIT/GCE at different scan speeds. The sweep rate was changed from 20 mV·s −1 to 200 mV·s −1 , and the redox peak current value was found to be proportional to the square root of the scan speed ( Figure 5), indicating that the electrochemical process was controlled by diffusion. The peak-to-peak potential difference between the cyclic volt-ampere of the sweep speed from 40 mV·s −1 to 180 mV·s −1 showed that the average electron transfer rate parameter ks of EPC fixed in CHIT was 1.06 s −1 . This value was calculated according to the Laviron model using the formula ks = mnFv/RT. In the formula, n represents the electron transfer number; F represents the Faraday constant; m is the peak-to-peak potential difference parameter; v is the scanning speed; R is the gas constant; and T is the temperature, respectively [19]. Here, T = 298 K, n = 1. The ks in this study were smaller than the reported biosensors using enzymes as catalysts [20,21], which further demonstrates that EPC can promote electron transfer.   Figure 5 shows the electrochemical response of EPC/CHIT/GCE at different scan speeds. The sweep rate was changed from 20 mV·s −1 to 200 mV·s −1 , and the redox peak current value was found to be proportional to the square root of the scan speed ( Figure 5), indicating that the electrochemical process was controlled by diffusion. The peak-to-peak potential difference between the cyclic volt-ampere of the sweep speed from 40 mV·s −1 to 180 mV·s −1 showed that the average electron transfer rate parameter ks of EPC fixed in CHIT was 1.06 s −1 . This value was calculated according to the Laviron model using the formula ks = mnFv/RT. In the formula, n represents the electron transfer number; F represents the Faraday constant; m is the peak-to-peak potential difference parameter; v is the scanning speed; R is the gas constant; and T is the temperature, respectively [19]. Here, T = 298 K, n = 1. The ks in this study were smaller than the reported biosensors using enzymes as catalysts [20,21], which further demonstrates that EPC can promote electron transfer.  Figure 5 shows the electrochemical response of EPC/CHIT/GCE at different scan speeds. The sweep rate was changed from 20 mV·s −1 to 200 mV·s −1 , and the redox peak current value was found to be proportional to the square root of the scan speed ( Figure 5), indicating that the electrochemical process was controlled by diffusion. The peak-to-peak potential difference between the cyclic volt-ampere of the sweep speed from 40 mV·s −1 to 180 mV·s −1 showed that the average electron transfer rate parameter k s of EPC fixed in CHIT was 1.06 s −1 . This value was calculated according to the Laviron model using the formula k s = mnFv/RT. In the formula, n represents the electron transfer number; F represents the Faraday constant; m is the peak-to-peak potential difference parameter; v is the scanning speed; R is the gas constant; and T is the temperature, respectively [19]. Here, T = 298 K, n = 1. The k s in this study were smaller than the reported biosensors using enzymes as catalysts [20,21], which further demonstrates that EPC can promote electron transfer.

Sensor Response Characterization
In order to compare the electrocatalytic properties of EPC/CHIT/GCE with CHIT/GCE and bare GCE, the catalytic performance of three modified electrodes were investigated by the electrocatalytic reduction of H2O2. Figure 6 is a current-time (it) curve for three modified electrodes. When adding 10 μL of 0.1 M H2O2 to 10 mL of PBS containing 1 mM hydroquinone, CHIT/GCE and bare GCE had almost no apparent catalytic response (see lines a and b in Figure 6), whereas EPC/CHIT/GCE (see line c in Figure 6) showed a significant catalytic response to different concentrations of H2O2 with a typical current-time response curve. Experiments show that the electrochemical sensor had a fast and sensitive response to H2O2.

Sensor Response Characterization
In order to compare the electrocatalytic properties of EPC/CHIT/GCE with CHIT/GCE and bare GCE, the catalytic performance of three modified electrodes were investigated by the electrocatalytic reduction of H 2 O 2 . Figure 6 is a current-time (it) curve for three modified electrodes. When adding 10 µL of 0.1 M H 2 O 2 to 10 mL of PBS containing 1 mM hydroquinone, CHIT/GCE and bare GCE had almost no apparent catalytic response (see lines a and b in Figure 6), whereas EPC/CHIT/GCE (see line c in Figure 6) showed a significant catalytic response to different concentrations of H 2 O 2 with a typical current-time response curve. Experiments show that the electrochemical sensor had a fast and sensitive response to H 2 O 2 .

Sensor Response Characterization
In order to compare the electrocatalytic properties of EPC/CHIT/GCE with CHIT/GCE and bare GCE, the catalytic performance of three modified electrodes were investigated by the electrocatalytic reduction of H2O2. Figure 6 is a current-time (it) curve for three modified electrodes. When adding 10 μL of 0.1 M H2O2 to 10 mL of PBS containing 1 mM hydroquinone, CHIT/GCE and bare GCE had almost no apparent catalytic response (see lines a and b in Figure 6), whereas EPC/CHIT/GCE (see line c in Figure 6) showed a significant catalytic response to different concentrations of H2O2 with a typical current-time response curve. Experiments show that the electrochemical sensor had a fast and sensitive response to H2O2.  With a signal-to-noise ratio of 3 and a correlation coefficient of 0.998 (n = 15), the linear detection range for H 2 O 2 ranged from 15 µM to 1.6 mM with a minimum detection limit of 3.7 µM and sensitivity theory for EPC/CHIT/GCE. The calculated value was 204.5 µA·mM −1 ·cm −2 . Table 1 summarizes the analytical parameters for the most relevant non-enzymatic hydrogen peroxide sensors based on the use of different metallic nanoparticles reported since 2013. The comparison allowed us to conclude that our sensor presented a comparable [22][23][24][25] linear range and sensitivity to most of the sensors. However, our sensor presented the great advantage of easy preparation and no precious metals. The Michaelis constant (K m ) is a kinetic parameter for studying the binding catalysis of an enzyme or catalyst to a substrate. In general, a smaller K m indicates a higher affinity of the enzyme or catalyst to the substrate. This value can be calculated using the Lineweaver-Burk equation (Equation (1)) [26].
In this equation, I ss is the steady state current value after substrate addition; I max is the maximum current at substrate concentration saturation; and C is the substrate concentration. In this work, the calculated value was 3.67 mM (see insert diagram (bottom), Figure 7). This value was lower than the 8.01 mM based on the HRP-ZrO 2 nanocomposite sensor [27] and much higher than the 41 mM based on the Nafion-Hb-CNT sensor [26]. Comparison of the surface results, especially with enzyme sensing, showed that EPC had higher enzymatic activity and had a good affinity for H 2 O 2 .

Sensor Selectivity, Reproducibility and Stability
Several possible interfering substances were selected to examine the selectivity of the sensor. The experiment was to compare the response current of the sensor to 0.1 mM H2O2 in the presence or

Sensor Selectivity, Reproducibility and Stability
Several possible interfering substances were selected to examine the selectivity of the sensor. The experiment was to compare the response current of the sensor to 0.1 mM H 2 O 2 in the presence or absence of 1.0 mM interfering substances. The experimental results (  To further determine the performance of this non-enzymatic sensor, we investigated its operational repeatability and long-term stability. The operational stability of the 2 mM H 2 O 2 electrocatalyst for 7 h was investigated by the same EPC/CHIT/GCE, and the results showed no significant change or difference. The standard deviation of H 2 O 2 at the same concentration for ten consecutive times was only 0.8%, indicating that the sensor has good repeatability. In order to evaluate the repeatability between the different modified electrodes, the standard deviation of the response of the five separately prepared electrodes to the same concentration of H 2 O 2 under the same conditions was only 2.3%, indicating that the electrodes still had high repeatability, which further explains the sensor with high practicality. The long-term stability was also systematically studied, and the same sensor was used to detect the same concentration of H 2 O 2 every few days. After one month of research, the data showed no significant change, which means that the sensor had high long-term stability. The most critical reason for achieving such high repeatability and stability is that the EPC does not undergo denaturation or reduced activity like other proteins or enzymes, and the physicochemical properties of the material are extremely stable.

Possible Response Mechanism
Based on the above experimental results, it is speculated that the possible mechanism of the non-enzymatic sensor's response to hydrogen peroxide is as follows: eucalyptus porous carbon (Fe 2+ ) + H 2 O 2 + H + → eucalyptus porous carbon (Fe 3+ ) + H 2 O eucalyptus porous carbon (Fe 3+ ) + H 2 Q → eucalyptus porous carbon (Fe 2+ ) + Q + H + Among them, H 2 Q and BQ represent the electron mediator hydroquinone and its oxidized form (p-benzoquinone), respectively. Under the action of hydroquinone, two electron transfers occurred first, H 2 O 2 was reduced to water, and the divalent iron ions in the porous carbon of eucalyptus were oxidized to ferric ions, while ferric ions in the eucalyptus porous carbon were reduced to divalent iron ions by hydroquinone. Subsequently, the p-benzoquinone rapidly acquired two electrons from the electrode and was reduced to hydroquinone. The peak current in the system is therefore related to the concentration of H 2 O 2 in the solution.

Conclusions
In this paper, a porous carbon material with biological morphology was prepared by a simple and effective carbonization technique. The prepared EPC had high electrocatalytic activity and affinity for H 2 O 2 with the aid of hydroquinone. The non-enzyme electrochemical sensors prepared using the ordered porous material had high sensitivity, high accuracy, high repeatability and stability. In addition,