# A Novel Nonenzymatic Hydrogen Peroxide Sensor Based on Magnetic Core-Shell Fe3O4@C/Au Nanoparticle Nanocomposite

Authors: Xiao Ni; Mingwei Tian; Jun Sun; Xiaojun Chen
Journal: International Journal of Analytical Chemistry
 (2021)

---

## Abstract

Fe3O4@C/Au nanoparticle (AuNP) nanocomposites were prepared through electrostatic adsorption of AuNPs onto PDDA-functionalized core/shell Fe3O4@C magnetic nanospheres, which had been synthesized by a facile solvothermal method. The morphology and composition of the nanocomposites were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR), etc. Moreover, highly electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) was also exhibited on the Fe3O4@C/AuNP-modified indium tin oxide (ITO) electrode. The effect of solution pH and the modification amount of Fe3O4@C/AuNPs on the performance of electrocatalytic H2O2 reduction was investigated. Under the optimal conditions, the catalytic current showed a linear relationship with the increase of H2O2 concentration in the range of 0.007–15 mM and a detection limit of 5 μM. The H2O2 sensor showed high selectivity for H2O2 detection, which could effectively resist the interference of ascorbic acid (AA), uric acid (UA), and citric acid (CA). Finally, the H2O2 sensor was used in the real fetal bovine serum to detect H2O2 and obtained satisfactory results with the recovery values ranging from 95.14 to 103.6%.


---

## Body

## 1. Introduction

Magnetic nanoparticles have been popularly applied in anode material [1], degradation [2], electromagnetic wave absorption [3], separation [4], and catalysis [5]. Among these diverse magnetic nanomaterials reported before, Fe3O4 NPs have been studied extensively because of their excellent magnetism, biocompatibility, and especially, highly effective electrocatalytic activity. For example, Kingsley et al. fabricated Fe3O4 nanoparticle- (NP-) modified carbon paste electrode to detect ascorbic acid (AA) and folic acid (FA) at the same time, with the detection limits of 2.51 × 10–7 and 2.01 × 10–9 M, respectively [6]. Qu and coworkers established a novel hydrogen peroxide (H2O2) sensor using Fe3O4 NPs/chitosan composite-modified electrode, which could electrocatalyze the reduction of H2O2 with a detection limit of 1.53 × 10–6 M [7]. However, Fe3O4 NPs are unstable and prone to be aggregated and settled in aqueous solution because of the magnetic dipolar-dipolar attraction, resulting in the decrease of their catalytic activity [8]. To overcome this problem, considerable efforts have been devoted to introduce stabilizers on the surface of Fe3O4 NPs.In recent years, core/shell structured magnetic nanocomposites have been proposed, with Fe3O4 NPs as the core and inorganic materials or polymers as the shell. These specific core/shell Fe3O4 materials possess enhanced stability and compatibility with different charges, functions, or reactive moieties. Owing to the enlarged effective surface area and the synergetic interaction between core and shell, these nanocomposites usually combine the advantageous properties of both core and shell [9]. For example, Beitollahi and coworkers synthesized a carbon paste electrode modified with magnetic Fe3O4@SiO2/MWCNT nanocomposite and found it exhibiting a very good resolution between the voltammetric peaks of hydrazine and phenol, which made it suitable for the detection of hydrazine in the presence of phenol in real samples [10]. Wang and coworkers showed that the Fe3O4@ZnO nanoparticles (NPs) have excellent photocatalytic activity and it is convenient to reuse NPs in wastewater treatment owing to the magnetic Fe3O4 core [11]. Therefore, magnetic core/shell nanocomposites with multiple properties have been widely used.Among these shell materials, carbon and gold are the most applied coating materials due to their excellent conductivity and biocompatibility [12, 13]. In addition, on the surface of carbon shells, abundant amounts of hydroxyl (-OH) and carboxyl (-COOH) groups can effectively protect the Fe3O4 NPs from aggregation and facilitate further functional modification over the surface. Hence, the applicability of Fe3O4@C has been extensively promoted. For example, Zhang and coworkers synthesized Fe3O4@C NPs, which showed dramatic ability to remove methylene blue, with a maximum adsorption of 141.3 mg·g−1 [14]. Zhang and coworkers fabricated another Fe3O4@C NPs immobilized upon the GCE which was modified with molecular imprinted TiO2 velamen. It was then made into the uric acid sensor, which delivered a linear range of 0.3–34 μM with a detection limit of 0.02 μM [15]. Meanwhile, functionalized Fe3O4@Au NPs have also been applied in As (III) [16] and dopamine [17] sensors, respectively.In this work, Fe3O4@C core/shell nanocomposite was synthesized by a facile solvothermal method from ferrocene as the raw material. It was not only easily separated by an external magnetic field but also possessed excellent dispersion in water. To further enhance the conductivity and catalytic activity, Au NPs were adsorbed on the surface of the obtained Fe3O4@C nanospheres by electrostatic attraction. The synthesized Fe3O4@C/Au NPs were first used to fabricate a nonenzymatic H2O2 sensor and showed high electrochemical sensitivity.


## 2. Materials and Methods

### 2.1. Materials

Ferrocene, acetone, sodium citrate, absolute ethanol, sodium chloride (NaCl), and Tris base were purchased from Shanghai Lingfeng Chemical Reagent (China). Poly(diallyldimethylammonium chloride) (PDDA, 20 wt. %) was purchased from Aldrich. Chloroauric acid hydrated (HAuCl4·4H2O) and sodium citrate were purchased from Nanjing Chemical Reagent (China). H2O2 (30%) was obtained from Sinopharm Chemical Reagent. Phosphate buffer saline (PBS, 0.1 M) was prepared by mixing the stock solutions of KH2PO4 and K2HPO4 and was adjusted to the appropriate pH value. Uric acid (UA), AA, and citric acid (CA) were all purchased from Sigma. Nafion (Nf) was obtained from DuPont as 5 wt% solution and was used as 0.5 wt% solution after dilution with water. Other reagents were of analytical grade and used without further purification. Double distilled water (DDW) was used throughout the experiments.


### 2.2. Characterization

The structures and morphologies of the Fe3O4@C and Fe3O4@C/AuNPs were characterized by transmission electron microscopy (TEM, JEOL JEM-200CX) and field emission scanning electron microscopy (FESEM, Hitachi S4800). Fourier transform infrared (FT-IR) spectra of KBr powder-pressed pellets were recorded on a Bruker model VECTOR 22 Fourier transform spectrometer. Elemental composition and chemical valence analyses were performed on X-ray diffraction (XRD, Rigaku SmartLab) and X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha), respectively.Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were performed using a CHI 660D electrochemical workstation (Shanghai Chenhua Instruments, China). A three-compartment electrochemical cell contained a saturated calomel reference electrode (SCE), a platinum wire auxiliary electrode, and a modified ITO working electrode with a 3 mm diameter controlled area. The determination of H2O2 was conducted in 0.1 M PBS, and the sensor responses were measured under deductions for background currents from the total ones.


### 2.3. Preparation of Fe3O4@C/AuNP Magnetic Nanocomposite

Core/shell structured Fe3O4@C magnetic nanocomposite was prepared by Wang’s method [18]. First, 0.3 g of ferrocene was dissolved in 30 mL of acetone under ultrasonication at room temperature for 30 min. Then, 1 mL of H2O2 was added dropwise into the above ferrocene solution. After reacting for 15 min under stirring, the resulting solution was transferred into a Teflon-lined stainless-steel autoclave with a capacity of 50 mL and heated at 210°C for 24 h. After cooling to room temperature, the product was magnetically separated, washed several times with absolute ethanol, and finally dried under vacuum at 50°C for 5 h.Au colloid solution was prepared by adding 5 mL of 1% (wt. %) sodium citrate into 200 mL of 0.01% (wt. %) HAuCl4, which had been heated to boil beforehand. Heating was stopped by the change of solution color from light blue to violet-red and stirring was continued for 15 min. After cooling to room temperature, Au colloid solution was preserved in a refrigerator at 4°C. Generally, an electrostatic attraction method was used for the immobilization of AuNPs onto the surface of the Fe3O4@C core/shell nanocomposite. 50 mg of Fe3O4@C nanocomposite was dispersed into 30 mL of 0.2% PDDA that contained 20 mM Tris base and 20 mM NaCl, and the mixture was stirred for 20 min. The product was collected with the help of a magnet and rinsed with DDW six times to remove the residual PDDA. The positively charged Fe3O4@C nanocomposite was dispersed in 100 mL of Au colloid solution and stirred for 8 h. The suspension was separated with the help of a magnet. After rinsed with water and ethanol three times successively, the Fe3O4@C/AuNPs nanocomposite was dried under vacuum at 50°C for 6 h.


### 2.4. Fabrication of Fe3O4@C/AuNP-Modified ITO Magnetism Electrode

Before modification, the ITO chips were cleaned with acetone, ethanol, and water, respectively, and then dried under a stream of nitrogen. The active electrode area was confined by bounding a circular opened 3M polyvinyl chloride (PVC) film on the ITO surface, with a 3 mm diameter on the middle of the electrode. On the other side of the ITO, a piece of 8 mm diameter and 5 mm height permanent magnet was adhered by silica rubber adhesive [19]. Then, 10 µL of 1 mg·mL−1 Fe3O4@C/AuNP nanocomposite dispersion (in 5 mL 0.5% Nf) was dropped onto the surface of the ITO magnetism electrode and dried at room temperature, which was designated as Fe3O4@C/AuNPs/Nf/ITO. When the sensor was not in use, it was stored at 4°C in a refrigerator.


## 2.1. Materials

Ferrocene, acetone, sodium citrate, absolute ethanol, sodium chloride (NaCl), and Tris base were purchased from Shanghai Lingfeng Chemical Reagent (China). Poly(diallyldimethylammonium chloride) (PDDA, 20 wt. %) was purchased from Aldrich. Chloroauric acid hydrated (HAuCl4·4H2O) and sodium citrate were purchased from Nanjing Chemical Reagent (China). H2O2 (30%) was obtained from Sinopharm Chemical Reagent. Phosphate buffer saline (PBS, 0.1 M) was prepared by mixing the stock solutions of KH2PO4 and K2HPO4 and was adjusted to the appropriate pH value. Uric acid (UA), AA, and citric acid (CA) were all purchased from Sigma. Nafion (Nf) was obtained from DuPont as 5 wt% solution and was used as 0.5 wt% solution after dilution with water. Other reagents were of analytical grade and used without further purification. Double distilled water (DDW) was used throughout the experiments.


## 2.2. Characterization

The structures and morphologies of the Fe3O4@C and Fe3O4@C/AuNPs were characterized by transmission electron microscopy (TEM, JEOL JEM-200CX) and field emission scanning electron microscopy (FESEM, Hitachi S4800). Fourier transform infrared (FT-IR) spectra of KBr powder-pressed pellets were recorded on a Bruker model VECTOR 22 Fourier transform spectrometer. Elemental composition and chemical valence analyses were performed on X-ray diffraction (XRD, Rigaku SmartLab) and X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha), respectively.Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were performed using a CHI 660D electrochemical workstation (Shanghai Chenhua Instruments, China). A three-compartment electrochemical cell contained a saturated calomel reference electrode (SCE), a platinum wire auxiliary electrode, and a modified ITO working electrode with a 3 mm diameter controlled area. The determination of H2O2 was conducted in 0.1 M PBS, and the sensor responses were measured under deductions for background currents from the total ones.


## 2.3. Preparation of Fe3O4@C/AuNP Magnetic Nanocomposite

Core/shell structured Fe3O4@C magnetic nanocomposite was prepared by Wang’s method [18]. First, 0.3 g of ferrocene was dissolved in 30 mL of acetone under ultrasonication at room temperature for 30 min. Then, 1 mL of H2O2 was added dropwise into the above ferrocene solution. After reacting for 15 min under stirring, the resulting solution was transferred into a Teflon-lined stainless-steel autoclave with a capacity of 50 mL and heated at 210°C for 24 h. After cooling to room temperature, the product was magnetically separated, washed several times with absolute ethanol, and finally dried under vacuum at 50°C for 5 h.Au colloid solution was prepared by adding 5 mL of 1% (wt. %) sodium citrate into 200 mL of 0.01% (wt. %) HAuCl4, which had been heated to boil beforehand. Heating was stopped by the change of solution color from light blue to violet-red and stirring was continued for 15 min. After cooling to room temperature, Au colloid solution was preserved in a refrigerator at 4°C. Generally, an electrostatic attraction method was used for the immobilization of AuNPs onto the surface of the Fe3O4@C core/shell nanocomposite. 50 mg of Fe3O4@C nanocomposite was dispersed into 30 mL of 0.2% PDDA that contained 20 mM Tris base and 20 mM NaCl, and the mixture was stirred for 20 min. The product was collected with the help of a magnet and rinsed with DDW six times to remove the residual PDDA. The positively charged Fe3O4@C nanocomposite was dispersed in 100 mL of Au colloid solution and stirred for 8 h. The suspension was separated with the help of a magnet. After rinsed with water and ethanol three times successively, the Fe3O4@C/AuNPs nanocomposite was dried under vacuum at 50°C for 6 h.


## 2.4. Fabrication of Fe3O4@C/AuNP-Modified ITO Magnetism Electrode

Before modification, the ITO chips were cleaned with acetone, ethanol, and water, respectively, and then dried under a stream of nitrogen. The active electrode area was confined by bounding a circular opened 3M polyvinyl chloride (PVC) film on the ITO surface, with a 3 mm diameter on the middle of the electrode. On the other side of the ITO, a piece of 8 mm diameter and 5 mm height permanent magnet was adhered by silica rubber adhesive [19]. Then, 10 µL of 1 mg·mL−1 Fe3O4@C/AuNP nanocomposite dispersion (in 5 mL 0.5% Nf) was dropped onto the surface of the ITO magnetism electrode and dried at room temperature, which was designated as Fe3O4@C/AuNPs/Nf/ITO. When the sensor was not in use, it was stored at 4°C in a refrigerator.


## 3. Results and Discussion

### 3.1. Structure and Composition Characterization of Samples

The structure and morphology of the nanocomposites were investigated by TEM and FESEM. In Figure1(a), Fe3O4@C with obvious core/shell structures were successfully synthesized. The presence of carbon shells prevented agglomeration of the Fe3O4 NPs and improved the dispersity of the Fe3O4@C, with an average diameter of approximately 125 nm. As vividly shown in Figure 1(b), the SEM image of Fe3O4@C displays a distinct margin without agglomeration. Figure 1(c) shows the TEM image of Fe3O4@C/AuNPs with a diameter of 141 nm. It is clear that there are numerous dark spots embedded in the outer shells, indicating that AuNPs were successfully immobilized on the surface of the Fe3O4@C nanospheres, with an average diameter of approximately 15 nm. Figure 1(d) shows the SEM image of the rather rough surface of Fe3O4@C/AuNPs with a raspberry-shaped morphology compared to that of Fe3O4@C. Moreover, some of them are overlapped or amalgamated due to external immobilization of AuNPs, which amplify the superficial roughness. And the dispersity of the Fe3O4@C nanocomposites is weakened by electrostatic attraction between AuNPs and Fe3O4@C as well. The inset in Figure 1(d) shows the elemental composition of Fe3O4@C/AuNPs and the Au atom mass concentration is 3.57%.Figure 1
(a, c) TEM and (b, d) FESEM images of Fe3O4@C and Fe3O4@C/AuNPs. The inset shows the elemental composition of Fe3O4@C/AuNPs.
(a)(b)(c)(d)FT-IR was used to characterize the chemical structure of the samples. Figure2(a) shows the FT-IR spectra of Fe3O4 (curve a), Fe3O4@C (curve b), and Fe3O4@C/AuNPs (curve c). The characteristic peak appears at 590 cm-1 which is related to the Fe-O stretching vibration [20]. Compared with curve a, there is a new strong band in curve b at around 3446 cm-1, indicating the presence of –OH in the carbon shell. These phenomena further proved that the carbon shell is successfully coated on the surface of the Fe3O4 nanosphere. The peak located at 1399 cm−1 is attributed to the COO- symmetric vibration [21]. Actually, the carboxyl group-functionalized Fe3O4@C nanospheres are negatively charged. This is the reason why we use PDDA to make the surface of carbon shell positively charged for the assembly of negatively charged AuNPs via electrostatic interaction. Curve c shows the FT-IR spectrum of Fe3O4@C/AuNPs. Because AuNPs do not have absorption in the IR region, the characteristic peak of Fe3O4@C/AuNPs is almost the same as that of Fe3O4@C.Figure 2
(a) FT-IR spectra of the Fe3O4 (curve a), Fe3O4@C (curve b), and Fe3O4@C/AuNPs (curve c), respectively, (b) XRD spectra of the Fe3O4@C/AuNPs. XPS spectra of (c) Fe 2p and (d) Au 4f from Fe3O4@C/AuNPs.
(a)(b)(c)(d)To characterize the crystal information of the prepared Fe3O4@C/AuNPs further, the XRD has been performed. As shown in Figure 2(b), the diffraction peaks appeared at 30°, 35.5°, 43°, 57°, and 62°are originated from Fe3O4, corresponding to (220), (311), (400), (511), and (440) crystal planes [22]. In addition to those peaks, another peak at 38.2° is in good accordance with (111) plane of AuNPs. It is difficult to discriminate Fe3O4 phases from γ-Fe2O3 by only using XRD patterns due to the similarity of patterns, and XPS is conducted to characterize the electronic structures of Fe element in the nanocomposite, which is known to be sensitive to Fe2+ and Fe3+ cations. Figure 2(c) presents two peaks at the binding energies of 724.4 and 711.4 eV, which correspond to the maximum intensity of Fe 2p1/2 and Fe 2p3/2, illustrating the coexistence of Fe2+ and Fe3+ cations in Fe3O4@C/AuNPs [23]. Peaks of Au are also shown in Figure 2(d), corresponding to Au 4f2/7 and Au 4f2/5, respectively. From the above characterization results, the well-defined core/shell structured Fe3O4@C/AuNPs is confirmed to be constructed.


### 3.2. Characterization of Nanocomposite-Modified ITO Electrodes

The influence of potential scan rate (v) on the response of the Fe3O4@C/AuNP-modified ITO electrode in PBS was studied, as shown in Figure 3(a). The reduction and oxidation peak currents (ipa and ipc) increased linearly with the square root of scan rate (v1/2) accelerating from 40 to 300 mV·s-1, indicating a surface-controlled electrochemical process.Figure 3
(a) CVs of Fe3O4@C/AuNPs/ITO at the scan rates of 40, 60, 100, 150, 200, 250, and 300 mV·s−1 (from internal to external) in 0.1 M pH 7.4 PBS containing 0.1 mM H2O2, respectively. The inset shows the linear plots of the peak current vs. v1/2; (b) CVs of different modified electrodes: bare ITO (curve a), Fe3O4@C/ITO (curve b), and Fe3O4@C/AuNPs/ITO (curve c) in 0.1 M pH 7.4 PBS containing 0.1 mM H2O2 at a scan rate of 100 mV·s−1.
(a)(b)As shown in Figure3(b), we also recorded the CVs of various modified electrodes towards 0.1 mM H2O2 in 0.1 M pH 7.4 PBS at a scan rate of 100 mV·s-1. At the bare ITO, almost no peak of H2O2 is observed (curve a), except for a slight reduction peak. After the electrode was modified with Fe3O4@C nanocomposites, a small increase in ipa was observed (curve b), indicating the electrocatalytic reduction activity toward H2O2 possessed by Fe3O4@C nanocomposites. The reduction peak potential was around −0.35 V, similar to another reported nonenzymatic H2O2 sensor [24] and the ipa was 1.7 times higher compared with that in curve a. When the Fe3O4@C/AuNPs were assembled on the electrode, the peak current significantly increased (curve c) and the ipa is 2.4 times that of curve b, demonstrating that the AuNPs significantly improve the catalytic activity of Fe3O4@C nanocomposites.The electron transfer rate and the interfacial resistance, as shown in Figures4(a) and 4(b), respectively, can reflect the electrochemical properties. In Figure 4(a), obvious oxidation and reduction peaks were observed on both Fe3O4@C- and Fe3O4@C/AuNP-modified electrodes, belonging to different valences of Fe element. An increased peak current was found after surface modification with AuNPs, indicating that noble metals could promote electron transfer on the electrode. In addition, the electroactive surface area (ESA) was calculated to be 1.628 cm2 by Randles–Sevcik equation, which is nearly 23 times the geometric area. It means that the modification of Fe3O4@C/AuNPs increased the roughness of the electrode surface greatly, which could facilitate the absorption, transmission, and catalysis of H2O2.Figure 4
(a) CV and (b) EIS curves of Fe3O4@C/AuNPs/ITO (curve a) and Fe3O4@C/ITO (curve b) in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3−/4−.
(a)(b)Similar results were also confirmed by EIS measurements, as shown in Figure4(b). The diameter of the Nyquist curve revealed charge transfer resistance of the electrode (Rct), and a larger diameter means a greater resistance. Curve a presented a smaller Rct value at Fe3O4@C/AuNP-modified ITO, verifying AuNPs could enhance charge transfer significantly, compared with that in curve b.


### 3.3. Optimization of Experimental Parameters

The variations of oxidation-reduction potential of H2O2 in different pH values indicate diverse oxidation ability of H2O2, resulting in different sensitivities of sensors under different pH circumstances. The effect of solution pH on the electrocatalytic reduction of H2O2 was investigated. Figure 5(a) shows the linear plots of the reduction peak current versus the concentration of H2O2 from 0.1 to 1 mM over the pH range from 6 to 9. The electrocatalytic activity and sensitivity were much higher at the pH value of 7–8 and lower at 6 and 8. The possible reason was that Fe3O4 core was expected to dissolve in acidic circumstances and the catalytic activity faded. Meanwhile, H2O2 is prone to lose oxidizing ability with solution alkalinity increasing. Therefore, a nearly neutral condition is suitable for H2O2 detection and the PBS with physiologic pH value of human being which is about 7.4 was chosen as the supporting electrolyte.Figure 5
(a) Linear plots of the reduction peak current versus the concentration of H2O2 from 0.1 to 1 mM over the pH range from 6 to 9 at Fe3O4@C/AuNPs/ITO electrode; (b) dependence of reduction peak current of 1 mM H2O2 in 0.1 M pH 7.4 PBS upon the modification amount of Fe3O4@C/AuNPs.
(a)(b)Figure5(b) exhibits reduction peak current versus different modification amounts of Fe3O4@C/AuNPs. The response current towards 1 mM H2O2 rose along with the modification amount of Fe3O4@C/AuNPs increasing from 0.6 to 1 mg·mL-1 and reached a plateau when the modification amount exceeded 1 mg mL-1. The response speed was supposed to be delayed by thickening modification, which brought hidden troubles of its falling off from the electrode surface. It may lead to a decreased stability of the modified electrode and the smaller response current. In this way, 1 mg·mL-1 of Fe3O4@C/AuNPs was regarded to be the optimal amount of modification for H2O2 detection.


### 3.4. Amperometric Response of H2O2 Sensor

Figure6 displays CVs in the presence of H2O2 at different concentrations under the optimum conditions. The response current shows a good linear relationship with the increase of H2O2 concentration in the range of 0.007–15 mM. The linear regression equations is ∆ip (µA) = 0.10 + 2.01 c (H2O2) (mM) (R2 = 0.9988). The detection limit was estimated at 5 µM, and the theoretical sensitivity of this sensor was calculated to be 28.71 µA mM−1 cm−2. The response current became saturated when the concentration of H2O2 exceeded 15 mM. Quantitative detection of H2O2 concentration can be realized by the calculation with the linear equation. Furthermore, the performances including electrode modification, linear range, and detection limit of the Fe3O4@C/AuNPs/ITO were compared with those of other H2O2 sensors, which are listed in Table 1. It was shown that the proposed H2O2 sensor in this work exhibited a wider linear range and a relatively lower detection limit. In comparison with those previous works, the advantages of our work are ascribed to five aspects: (1) the combination of the magnetic core of Fe3O4, thin layer of C, and uniformly dispersed AuNP modification shows their synergies and thus enhances the catalytic ability towards H2O2; (2) the C layer prevents Fe3O4 catalyst from aggregating and maintains its catalytic activity; (3) the utilization of magnetic electrode increases the modification amount of Fe3O4@C/AuNPs and enhances the stability of the electrode modification; (4) the C layer is not compact, which facilitate H2O2 and the reaction products transferring between the bulk solution and electrode surface; and (5) the modification of AuNPs enhances the electron transfer and electrochemical activity of the H2O2 sensor.Figure 6
(a) CVs of Fe3O4@C/AuNPs/ITO recorded in 0.1 M pH 7.4 PBS containing 0.007–15 mM of H2O2 (curves a to m, at a scan rate of 100 mV·s−1. The inset shows the CVs corresponding to the H2O2 concentration in the range of 0.007–1 mM (from a to h); (b) the calibration plot of the reduction peak current vs. the H2O2 concentration.
(a)(b)Table 1
The performance comparison of different nonenzymatic H2O2 sensors.
Electrode modificationLinear range (mM)Detection limit (μM)Ref.Co3O4/MWCNTs0.02–0.432.46[25]PtAu0.05–1.154.1[26]Pd/CNT0.008–9.52.6[27]AgCo/MWCNTs0.05–100.74[28]NPG@Ni0.02–9.7410[29]Se/PtNPs0.01–1.53.1[30]Ag/NaX0.02–11.769.1[31]Ag NW array0.1–3.129.2[32]AgNPs0.05–6.527[33]Fe3O4@C/CuNPs0.08–37232.6[34]Fe3O4@C/AuNPs0.007–155This work


### 3.5. Stability and Repeatability

The application of chemical sensors put forward higher demands on stability and catalytic signal repeatability. DPV and EIS measurements for Fe3O4@C/AuNP-modified ITO were conducted in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3-/4-. As shown in Figure 7, it was found that the DPV peak remained nearly unchanged after 10 cycles of scanning. On the other hand, the Nyquist plots nearly overlapped for 10 times of detection, and the Rct value maintained around 152 Ω. It demonstrated that the immobilized Fe3O4@C/AuNP modification layer could keep a good level of stability in a continuous scanning system.Figure 7
(a) DPV and (b) EIS curves of Fe3O4@C/AuNPs/ITO in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3-/4- with 10 continuous cycles of scanning. (c) The reduction peak current of 1 mM H2O2 in 0.1 M pH 7.4 PBS recorded on 5 parallel sensors.
(a)(b)(c)In addition, a repeated measurement towards 1 mM H2O2 in 0.1 M pH 7.4 PBS was conducted to verify the repeatability of the sensor. The response signal recorded on 5 parallel electrodes is displayed in Figure 7(c), and the relative standard deviation (RSD) value between them was calculated as 1.8%. The good stability and repeatability of the fabricated H2O2 sensor are attributed to the simple modification method, stable property, and good catalytic ability of Fe3O4@C/AuNPs.


### 3.6. Interference Study

To evaluate the selectivity of Fe3O4@C/AuNPs, an interference study was carried out. As it is known, AA, UA, and CA are the most common interfering electroactive species during the detection of H2O2. The current responses with the addition of H2O2, AA, UA, and CA at the potential of −0.35 V are shown in Figure 8, among which the responses caused by AA, UA, and CA interfering species were no more than 10% response of H2O2. The result implied that the Fe3O4@C/AuNPs possessed high selectivity for H2O2 detection at the potential of −0.35 V.Figure 8
Current responses after addition of 0.1 mM of H2O2, AA, UA, and CA at the potential of −0.35 V, respectively.


### 3.7. Determination of H2O2 in Fetal Bovine Serum (FBS)

The determination of H2O2 in FBS was also investigated. The FBS was diluted with 0.1 M of pH 7.4 PBS via the volume ratio of 1 : 9. We measured the recovery by adding different concentrations of H2O2 into the FBS dilution. The recovery values were calculated ranging from 95.14 to 103.6%, which are presented in Table 2. The relative standard deviations (RSDs) are less than 5%, proving a good application prospect.Table 2
The recovery of H2O2 by standard addition method in FBS samples.
SampleAdded concentration (mM)Measured concentration (mM)Recovery (%)10.0940.0995.1420.280.29103.630.470.48102.140.950.9498.9554.544.62101.8


## 3.1. Structure and Composition Characterization of Samples

The structure and morphology of the nanocomposites were investigated by TEM and FESEM. In Figure1(a), Fe3O4@C with obvious core/shell structures were successfully synthesized. The presence of carbon shells prevented agglomeration of the Fe3O4 NPs and improved the dispersity of the Fe3O4@C, with an average diameter of approximately 125 nm. As vividly shown in Figure 1(b), the SEM image of Fe3O4@C displays a distinct margin without agglomeration. Figure 1(c) shows the TEM image of Fe3O4@C/AuNPs with a diameter of 141 nm. It is clear that there are numerous dark spots embedded in the outer shells, indicating that AuNPs were successfully immobilized on the surface of the Fe3O4@C nanospheres, with an average diameter of approximately 15 nm. Figure 1(d) shows the SEM image of the rather rough surface of Fe3O4@C/AuNPs with a raspberry-shaped morphology compared to that of Fe3O4@C. Moreover, some of them are overlapped or amalgamated due to external immobilization of AuNPs, which amplify the superficial roughness. And the dispersity of the Fe3O4@C nanocomposites is weakened by electrostatic attraction between AuNPs and Fe3O4@C as well. The inset in Figure 1(d) shows the elemental composition of Fe3O4@C/AuNPs and the Au atom mass concentration is 3.57%.Figure 1
(a, c) TEM and (b, d) FESEM images of Fe3O4@C and Fe3O4@C/AuNPs. The inset shows the elemental composition of Fe3O4@C/AuNPs.
(a)(b)(c)(d)FT-IR was used to characterize the chemical structure of the samples. Figure2(a) shows the FT-IR spectra of Fe3O4 (curve a), Fe3O4@C (curve b), and Fe3O4@C/AuNPs (curve c). The characteristic peak appears at 590 cm-1 which is related to the Fe-O stretching vibration [20]. Compared with curve a, there is a new strong band in curve b at around 3446 cm-1, indicating the presence of –OH in the carbon shell. These phenomena further proved that the carbon shell is successfully coated on the surface of the Fe3O4 nanosphere. The peak located at 1399 cm−1 is attributed to the COO- symmetric vibration [21]. Actually, the carboxyl group-functionalized Fe3O4@C nanospheres are negatively charged. This is the reason why we use PDDA to make the surface of carbon shell positively charged for the assembly of negatively charged AuNPs via electrostatic interaction. Curve c shows the FT-IR spectrum of Fe3O4@C/AuNPs. Because AuNPs do not have absorption in the IR region, the characteristic peak of Fe3O4@C/AuNPs is almost the same as that of Fe3O4@C.Figure 2
(a) FT-IR spectra of the Fe3O4 (curve a), Fe3O4@C (curve b), and Fe3O4@C/AuNPs (curve c), respectively, (b) XRD spectra of the Fe3O4@C/AuNPs. XPS spectra of (c) Fe 2p and (d) Au 4f from Fe3O4@C/AuNPs.
(a)(b)(c)(d)To characterize the crystal information of the prepared Fe3O4@C/AuNPs further, the XRD has been performed. As shown in Figure 2(b), the diffraction peaks appeared at 30°, 35.5°, 43°, 57°, and 62°are originated from Fe3O4, corresponding to (220), (311), (400), (511), and (440) crystal planes [22]. In addition to those peaks, another peak at 38.2° is in good accordance with (111) plane of AuNPs. It is difficult to discriminate Fe3O4 phases from γ-Fe2O3 by only using XRD patterns due to the similarity of patterns, and XPS is conducted to characterize the electronic structures of Fe element in the nanocomposite, which is known to be sensitive to Fe2+ and Fe3+ cations. Figure 2(c) presents two peaks at the binding energies of 724.4 and 711.4 eV, which correspond to the maximum intensity of Fe 2p1/2 and Fe 2p3/2, illustrating the coexistence of Fe2+ and Fe3+ cations in Fe3O4@C/AuNPs [23]. Peaks of Au are also shown in Figure 2(d), corresponding to Au 4f2/7 and Au 4f2/5, respectively. From the above characterization results, the well-defined core/shell structured Fe3O4@C/AuNPs is confirmed to be constructed.


## 3.2. Characterization of Nanocomposite-Modified ITO Electrodes

The influence of potential scan rate (v) on the response of the Fe3O4@C/AuNP-modified ITO electrode in PBS was studied, as shown in Figure 3(a). The reduction and oxidation peak currents (ipa and ipc) increased linearly with the square root of scan rate (v1/2) accelerating from 40 to 300 mV·s-1, indicating a surface-controlled electrochemical process.Figure 3
(a) CVs of Fe3O4@C/AuNPs/ITO at the scan rates of 40, 60, 100, 150, 200, 250, and 300 mV·s−1 (from internal to external) in 0.1 M pH 7.4 PBS containing 0.1 mM H2O2, respectively. The inset shows the linear plots of the peak current vs. v1/2; (b) CVs of different modified electrodes: bare ITO (curve a), Fe3O4@C/ITO (curve b), and Fe3O4@C/AuNPs/ITO (curve c) in 0.1 M pH 7.4 PBS containing 0.1 mM H2O2 at a scan rate of 100 mV·s−1.
(a)(b)As shown in Figure3(b), we also recorded the CVs of various modified electrodes towards 0.1 mM H2O2 in 0.1 M pH 7.4 PBS at a scan rate of 100 mV·s-1. At the bare ITO, almost no peak of H2O2 is observed (curve a), except for a slight reduction peak. After the electrode was modified with Fe3O4@C nanocomposites, a small increase in ipa was observed (curve b), indicating the electrocatalytic reduction activity toward H2O2 possessed by Fe3O4@C nanocomposites. The reduction peak potential was around −0.35 V, similar to another reported nonenzymatic H2O2 sensor [24] and the ipa was 1.7 times higher compared with that in curve a. When the Fe3O4@C/AuNPs were assembled on the electrode, the peak current significantly increased (curve c) and the ipa is 2.4 times that of curve b, demonstrating that the AuNPs significantly improve the catalytic activity of Fe3O4@C nanocomposites.The electron transfer rate and the interfacial resistance, as shown in Figures4(a) and 4(b), respectively, can reflect the electrochemical properties. In Figure 4(a), obvious oxidation and reduction peaks were observed on both Fe3O4@C- and Fe3O4@C/AuNP-modified electrodes, belonging to different valences of Fe element. An increased peak current was found after surface modification with AuNPs, indicating that noble metals could promote electron transfer on the electrode. In addition, the electroactive surface area (ESA) was calculated to be 1.628 cm2 by Randles–Sevcik equation, which is nearly 23 times the geometric area. It means that the modification of Fe3O4@C/AuNPs increased the roughness of the electrode surface greatly, which could facilitate the absorption, transmission, and catalysis of H2O2.Figure 4
(a) CV and (b) EIS curves of Fe3O4@C/AuNPs/ITO (curve a) and Fe3O4@C/ITO (curve b) in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3−/4−.
(a)(b)Similar results were also confirmed by EIS measurements, as shown in Figure4(b). The diameter of the Nyquist curve revealed charge transfer resistance of the electrode (Rct), and a larger diameter means a greater resistance. Curve a presented a smaller Rct value at Fe3O4@C/AuNP-modified ITO, verifying AuNPs could enhance charge transfer significantly, compared with that in curve b.


## 3.3. Optimization of Experimental Parameters

The variations of oxidation-reduction potential of H2O2 in different pH values indicate diverse oxidation ability of H2O2, resulting in different sensitivities of sensors under different pH circumstances. The effect of solution pH on the electrocatalytic reduction of H2O2 was investigated. Figure 5(a) shows the linear plots of the reduction peak current versus the concentration of H2O2 from 0.1 to 1 mM over the pH range from 6 to 9. The electrocatalytic activity and sensitivity were much higher at the pH value of 7–8 and lower at 6 and 8. The possible reason was that Fe3O4 core was expected to dissolve in acidic circumstances and the catalytic activity faded. Meanwhile, H2O2 is prone to lose oxidizing ability with solution alkalinity increasing. Therefore, a nearly neutral condition is suitable for H2O2 detection and the PBS with physiologic pH value of human being which is about 7.4 was chosen as the supporting electrolyte.Figure 5
(a) Linear plots of the reduction peak current versus the concentration of H2O2 from 0.1 to 1 mM over the pH range from 6 to 9 at Fe3O4@C/AuNPs/ITO electrode; (b) dependence of reduction peak current of 1 mM H2O2 in 0.1 M pH 7.4 PBS upon the modification amount of Fe3O4@C/AuNPs.
(a)(b)Figure5(b) exhibits reduction peak current versus different modification amounts of Fe3O4@C/AuNPs. The response current towards 1 mM H2O2 rose along with the modification amount of Fe3O4@C/AuNPs increasing from 0.6 to 1 mg·mL-1 and reached a plateau when the modification amount exceeded 1 mg mL-1. The response speed was supposed to be delayed by thickening modification, which brought hidden troubles of its falling off from the electrode surface. It may lead to a decreased stability of the modified electrode and the smaller response current. In this way, 1 mg·mL-1 of Fe3O4@C/AuNPs was regarded to be the optimal amount of modification for H2O2 detection.


## 3.4. Amperometric Response of H2O2 Sensor

Figure6 displays CVs in the presence of H2O2 at different concentrations under the optimum conditions. The response current shows a good linear relationship with the increase of H2O2 concentration in the range of 0.007–15 mM. The linear regression equations is ∆ip (µA) = 0.10 + 2.01 c (H2O2) (mM) (R2 = 0.9988). The detection limit was estimated at 5 µM, and the theoretical sensitivity of this sensor was calculated to be 28.71 µA mM−1 cm−2. The response current became saturated when the concentration of H2O2 exceeded 15 mM. Quantitative detection of H2O2 concentration can be realized by the calculation with the linear equation. Furthermore, the performances including electrode modification, linear range, and detection limit of the Fe3O4@C/AuNPs/ITO were compared with those of other H2O2 sensors, which are listed in Table 1. It was shown that the proposed H2O2 sensor in this work exhibited a wider linear range and a relatively lower detection limit. In comparison with those previous works, the advantages of our work are ascribed to five aspects: (1) the combination of the magnetic core of Fe3O4, thin layer of C, and uniformly dispersed AuNP modification shows their synergies and thus enhances the catalytic ability towards H2O2; (2) the C layer prevents Fe3O4 catalyst from aggregating and maintains its catalytic activity; (3) the utilization of magnetic electrode increases the modification amount of Fe3O4@C/AuNPs and enhances the stability of the electrode modification; (4) the C layer is not compact, which facilitate H2O2 and the reaction products transferring between the bulk solution and electrode surface; and (5) the modification of AuNPs enhances the electron transfer and electrochemical activity of the H2O2 sensor.Figure 6
(a) CVs of Fe3O4@C/AuNPs/ITO recorded in 0.1 M pH 7.4 PBS containing 0.007–15 mM of H2O2 (curves a to m, at a scan rate of 100 mV·s−1. The inset shows the CVs corresponding to the H2O2 concentration in the range of 0.007–1 mM (from a to h); (b) the calibration plot of the reduction peak current vs. the H2O2 concentration.
(a)(b)Table 1
The performance comparison of different nonenzymatic H2O2 sensors.
Electrode modificationLinear range (mM)Detection limit (μM)Ref.Co3O4/MWCNTs0.02–0.432.46[25]PtAu0.05–1.154.1[26]Pd/CNT0.008–9.52.6[27]AgCo/MWCNTs0.05–100.74[28]NPG@Ni0.02–9.7410[29]Se/PtNPs0.01–1.53.1[30]Ag/NaX0.02–11.769.1[31]Ag NW array0.1–3.129.2[32]AgNPs0.05–6.527[33]Fe3O4@C/CuNPs0.08–37232.6[34]Fe3O4@C/AuNPs0.007–155This work


## 3.5. Stability and Repeatability

The application of chemical sensors put forward higher demands on stability and catalytic signal repeatability. DPV and EIS measurements for Fe3O4@C/AuNP-modified ITO were conducted in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3-/4-. As shown in Figure 7, it was found that the DPV peak remained nearly unchanged after 10 cycles of scanning. On the other hand, the Nyquist plots nearly overlapped for 10 times of detection, and the Rct value maintained around 152 Ω. It demonstrated that the immobilized Fe3O4@C/AuNP modification layer could keep a good level of stability in a continuous scanning system.Figure 7
(a) DPV and (b) EIS curves of Fe3O4@C/AuNPs/ITO in 0.1 M of pH 7.4 PBS containing 0.1 M KCl and 5 mM [Fe(CN)6]3-/4- with 10 continuous cycles of scanning. (c) The reduction peak current of 1 mM H2O2 in 0.1 M pH 7.4 PBS recorded on 5 parallel sensors.
(a)(b)(c)In addition, a repeated measurement towards 1 mM H2O2 in 0.1 M pH 7.4 PBS was conducted to verify the repeatability of the sensor. The response signal recorded on 5 parallel electrodes is displayed in Figure 7(c), and the relative standard deviation (RSD) value between them was calculated as 1.8%. The good stability and repeatability of the fabricated H2O2 sensor are attributed to the simple modification method, stable property, and good catalytic ability of Fe3O4@C/AuNPs.


## 3.6. Interference Study

To evaluate the selectivity of Fe3O4@C/AuNPs, an interference study was carried out. As it is known, AA, UA, and CA are the most common interfering electroactive species during the detection of H2O2. The current responses with the addition of H2O2, AA, UA, and CA at the potential of −0.35 V are shown in Figure 8, among which the responses caused by AA, UA, and CA interfering species were no more than 10% response of H2O2. The result implied that the Fe3O4@C/AuNPs possessed high selectivity for H2O2 detection at the potential of −0.35 V.Figure 8
Current responses after addition of 0.1 mM of H2O2, AA, UA, and CA at the potential of −0.35 V, respectively.


## 3.7. Determination of H2O2 in Fetal Bovine Serum (FBS)

The determination of H2O2 in FBS was also investigated. The FBS was diluted with 0.1 M of pH 7.4 PBS via the volume ratio of 1 : 9. We measured the recovery by adding different concentrations of H2O2 into the FBS dilution. The recovery values were calculated ranging from 95.14 to 103.6%, which are presented in Table 2. The relative standard deviations (RSDs) are less than 5%, proving a good application prospect.Table 2
The recovery of H2O2 by standard addition method in FBS samples.
SampleAdded concentration (mM)Measured concentration (mM)Recovery (%)10.0940.0995.1420.280.29103.630.470.48102.140.950.9498.9554.544.62101.8


## 4. Conclusion

In summary, a novel core-shell structured Fe3O4@C/AuNPs nanocomposite is successfully synthesized and utilized for fabricating a nonenzymatic H2O2 sensor, exhibiting a wider linear range from 0.007 to 15 mM and a lower detection limit of 5 μM with a sensitivity of 28.71 µA mM−1 cm−2. The advantages of the proposed sensor over the previous works are concluded as four points: (1) the superparamagnetic property of Fe3O4@C/AuNPs nanocomposite contributes to the construction of a magnetic electrode modification, enhancing the stability of the sensor greatly. (2) The protection of C/AuNPs bilayer ensures the monodispersity of Fe3O4 catalyst, maintaining its good catalytic activity for H2O2 reduction. (3) The C/AuNPs bilayer possesses hydrophilicity, biocompatibility, electroactivity, and conductivity, facilitating the substance transmission and electron transfer between bulk solution and electrode surface. (4) The detection of H2O2 in FBS illustrates high selectivity, displaying good potential for real applications. Of course, the fabricated Fe3O4@C/AuNPs nanocomposite could also be used as substrate or nanolabel in other sensing systems.



---

*Source: 8839895-2021-03-08.xml*