Development of an Amperometric Hydrogen Peroxide Biosensor based on the Immobilization of Horseradish Peroxidase onto Nickel Ferrite Nanoparticle-Chitosan Composite

Nickel ferrite (NiFe2O4) nanoparticles have been dispersed in chitosan solution in order to fabricate nanocomposite films. Horseradish peroxidase (HRP) has been immobilized onto this chitosan-NiFe2O4 nanocomposite film via physical adsorption. The size of the NiFe2O4 nanoparticles has been estimated using X-ray diffraction pattern and scanning electron microscopy (SEM) to be 40±9 nm. The chitosan-NiFe2O4 nanocomposite film and HRP/chitosan-NiFe2O4 bioelectrode have been characterized using SEM technique. The HRP/chitosan-NiFe2O4 nanocomposite bioelectrode has a response time of 4 s, linearity as 0.3 to 12 mM of H2O2, sensitivity as 22 nA/mM. The effects of pH and the temperature of the immobilized HRP electrode have also been studied.


Introduction
The rapid, accurate, reliable and reagentless sensing and detection of hydrogen peroxide is of great importance in textile/food and health care industries. Many techniques, including titrimetry [1], chemiluminescence [2], fluorescence [3] and spectrophotometry [4], tend to be complex, time-consuming and suffer from various interferences. Consequently, rapid and reliable methods for measuring H 2 O 2 are greatly sought after. Electrochemical methods, especially using various amperometric H 2 O 2 biosensors, have been extensively employed for the detection of H 2 O 2 . Several valuable efforts have been focused on the development of electrochemical biosensors based on horseradish peroxidase for the detection of H 2 O 2 . The performance of these biosen-sors depends on the methods of enzyme immobilization when fabricating enzyme-modified electrodes. Various immobilization techniques have been employed, including adsorption [5], cross-linking [6], layer-by-layer assembly [7], covalent binding [8], surfactant-enzyme complex formation [9], sol-gel entrapment [10], and the use of biological membranes [11] and nanomaterials [12].
Magnetic nanoparticles as special biomolecule immobilizing carriers are becoming the focus of research [13]. Magnetic nanoparticles have been used in biomedicine and immunology due to its special properties [14][15][16]. The applications of magnetic nanoparticles in the immobilization of biomolecules have been studied by Kaushik et al. [17] and Qu et al. [18]. Among magnetic nanoparticles, ferrites are a broad class of complex magnetic oxides of considerable technological importance [19,20]. Nickel ferrite (NiFe 2 O 4 ), with an inverse spinel structure shows good biocompatibility, noncytotoxicity, easy preparation and also ferromagnetism that originates from magnetic moment of anti-parallel spins between Fe 3+ ions at tetrahedral sites and Ni 2+ ions at octahedral sites [21,22]. Due to their large surface-tovolume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability, nanomaterials are utilized for biosensor applications. In a recent study, Luo et al. used chitosan/NiFe 2 O 4 nanocomposite as an enzyme immobilization matrix for glucose biosensor application [23]. This study shows that GOx incorporated chitosan/NiFe 2 O 4 nanocomposite is characteristic by an excellent electroanalytical response to glucose.
Due to the excellent film-forming ability, high permeability, mechanical strength, nontoxicity, biocompatibility, low cost and availability, chitosan has been found to be a workhorse biopolymer for the immobilization of biomolecules for biosensor applications. Much effort has been made to improve the performance of chitosan for application as biosensors. These adjustments include its structural modification, change in molecular factors, as well as the incorporation of metal oxide nanoparticles in the chitosan network structure [24,25].
The motivation of our work is to construct and analyze a simple biosensor using HRP for the detection of hydrogen peroxide. Optimized experimental conditions for the fabrication and operation of the biosensor have been established. The resulting biosensor has some advantages such as high sensitivity, good repeatability and reproducibility.

Materials and methods
Materials HRP (EC 1.11.1.7, RZ>3.0, 250 U/mg), and chitosan were obtained from Sigma. All other chemicals were of analytical grade and used without further purification.

Preparation of NiFe 2 O 4 nanoparticles
Nanoparticles of NiFe 2 O 4 were prepared through microwave-induced combustion.
Analytical grade 1 g NiCl 2 , 1 g Fe(NO 3 ) 2 , and with 1 g glycine (NH 2 CH 2 COOH), were dissolved in deionized water in the desired ratio. The crucible containing the solution was heated in a microwave oven (CEM, MDS 81D, 650 W). Initially, the solution boils and evaporates followed by decomposition with copious evolution of gases (N 2 , NO 2 , CO 2 ). When the mixture reaches the ignition temperature it begins burning and releases a great deal of heat from the highly exothermic reaction [26]. This vaporizes the remaining liquid instantly, and forms a solid burning at over 1000℃. The entire process takes only 15 min to produce ferrite powders. Glycine serves as fuel, being oxidized by nitrate. The evolution of large amounts of gases helps dissipate heat, reducing oxide sintering [27,28].

Preparation of NiFe 2 O 4 /Chitosan solution
Chitosan solution (0.5%) was obtained by dissolving 0.025 g of chitosan in 5 ml of 0.05 mol·l −1 acetate buffer solution (acetate dissolved in doubly distilled water). The calculated amount of NiFe 2 O 4 nanoparticles was dispersed in the chitosan solution by stirring at room temperature, followed by sonification. Finally, a highly viscous solution of chitosan with uniformly dispersed NiFe 2 O 4 nanoparticles was obtained.

Construction of HRP/NiFe 2 O 4 /chitosan biosensor
Before each experiment, the GCE (3 mm diameter) was first polished on chamois leather with 0.05 µm alumina powders and then washed ultrasonically in doubly distilled water, anhydrous ethanol, and water, respectively. After these pretreatments, the cleaned GCE was dried in air. HRP/NiFe 2 O 4 /chitosan solution was prepared by mixing HRP in NiFe 2 O 4 /chitosan solution. To get the best amperometric responses of the biosensor, the composition of the HRP/NiFe 2 O 4 /chitosan were optimized. 10 µl of solution was dropped on the surface of the cleaned GCE. Then the electrode was dried at room temperature. Before experiment, the biosensor was immersed in 0.1 mM PBS (pH=8) to wash out the non-immobilized components from the electrode surface. When not in use, the biosensor was preserved at 4℃ in a dry state.

Apparatus and measurements
X-ray powder diffraction (XRD) analysis was conducted on a Rigaku Smart Lab operated at 40 kV and 35 mA using Cu Kα radiation (λ=1.54059Å). FEI XL40 Sirion FEG Digital Scanning Microscope was used in order to investigate the microstructure and morphology of the sample. Samples were coated with gold at 10 mA for 2 min prior to the scanning electron microscopy (SEM) analysis.
All electrochemical measurements were performed using a CHI Model 842B electrochemical analyzer. These measurements were carried out using a three electrode cell with glassy carbon electrode as the working electrode, a platinum (Pt) wire as the counter electrode, and saturated Ag/AgCl electrode as a reference electrode in phosphate buffer saline (PBS, 10 mM, pH 8) containing [Fe(CN) 6 ] 3−/4− . All amperometric measurements were carried out at room temperature. They were performed in stirred solutions by applying the de-sired potential and allowing the steady state current to be reached. Once prepared, the enzyme electrode were immersed in 10 ml of a 10 mM PBS, pH 8 solution, and the amperometric responses to the addition of known amount of analyte solution were recorded, respectively. The data are the average of three measurements.
The porous film of chitosan contains pin holes as illustrated by the SEM micrograph in Fig. 2(a). Figure  2(b) shows that NiFe 2 O 4 nanoparticles are uniformly embedded in the porous CH network. The surface of  HRP immobilized NiFe 2 O 4 chitosan nanocomposite shows a homogeneous globular morphology (pocks due to the presence of HRP immobilized NiFe 2 O 4 in chitosan can be clearly observed) (Fig. 2(c)) revealing the immobilization of HRP.
Electrochemical behavior of HRP/NiFe 2 O 4 / chitosan modified electrode Electrochemical impedance spectroscopy (EIS) studies of chitosan film, NiFe 2 O 4 /chitosan and HRP/ NiFe 2 O 4 /chitosan bioelectrodes have been conducted in the frequency range of 0.01-10 5 Hz. In the EIS investigation, the semicircle part corresponds to the electron transfer limited process; its diameter is equal to the electron transfer resistance, R CT , which controls the electron transfer kinetics of the redox probe at the electrode interface. Figure     It can be seen that the anodic potential shifts towards positive side and the cathodic peak potential shifts in the reverse direction. Besides this, the redox peak currents are proportional to the square root of scan rate, υ 1/2 , indicating a diffusion electrontransfer process. it can be seen that the redox potential of NiFe 2 O 4 /chitosan electrode shifts towards the higher side than that of pure chitosan film due to the incorporation of NiFe 2 O 4 nanoparticles. It appears that the NiFe 2 O 4 /chitosan nanocomposite electrode provides a biocompatible environment to the HRP; and NiFe 2 O 4 nanoparticles act as an electron mediator resulting in an accelerated electron transfer between HRP and electrode [33]. The electrocatalytic behavior of HRP incorporated in the bionanocomposite has been evaluated by cyclic voltammetry. Since the proposed HRP electrode did not show direct electron transfer between immobilized HRP and GCE, K 4 Fe(CN) 6 was used as the electron mediator. Cyclic voltammograms (CVs) of the HRP/CS/GPTMS-modified electrode in 0.02 mol·l −1 PBS (pH=7.0) are shown in Fig. 5. When 40 µmol·l −1 H 2 O 2 was introduced, an obvious electrocatalytic response was observed with the increase of reduction current and the decrease of oxidation current. The response process of the biosensor may be expressed as follows:

Optimization experimental parameters
The amount of the enzyme in composite is a vital factor affecting the analytical sensitivity of the biosensor ( Fig. 6(a)). By the use of composite solutions containing different HRP concentrations and a fixed NiFe 2 O 4 concentration (2 mg·ml −1 ) for dropping on the surface of the GCE, the change of amperometric current with HRP amount under constant H 2 O 2 concentration is shown in Fig. 6. The current response increases as increasing HRP concentration and achieves a maximum value at 8.0 mg·ml −1 . With the increasing of HRP concentration from 8.0 to 10.0 mg·ml −1 , the sensitivity reduces gradually. So, an optimum loading of 8.0 mg·ml −1 HRP was used for subsequent experiments. Figure 6(b) shows the dependence of the current response of the modified electrode on the applied potential in the range from −0.3 V to 0 V under constant H 2 O 2 concentration. With applied potential decreasing from −0.1 V to 0 V, the steady state current increases due to the increased driving force for the fast reduction of H 2 O 2 at the lower potentials and approaches a maximum value at −0.1 V, then the response current decreases with applied potential decreasing from −0.3 V to −0.1 V. Therefore, −0.1 V was selected as the applied potential for amperometric measurement in subsequent experiments.
The effect of pH on the modified electrode response was investigated under constant H 2 O 2 concentration and the results are displayed in Fig. 6(c). The biosensor response increases with increasing pH value from 5.0 to 8.0, and achieves a maximum value at 8.0, then decreases from 8.0 to 9.0. So pH=8.0 PBS was chosen as the supporting electrolyte for the further experiments.
The activity of the enzyme electrode has been investigated using 15 mM H 2 O 2 solution in 10 mM PBS, pH=8.0 solution by amperometric measurements at temperature varying from 35 to 60℃, as shown in Fig. 6(d). It is observed that the response increases with rising temperature, reaching a maximum at 55℃, and then starts to decrease. This could have been caused by the denaturation of HRP or film instability at the higher temperatures.
Amperometric response of the biosensor to The amperometric measurement of H 2 O 2 at the enzyme electrode has been investigated and the calibration curve of the response current of the enzyme electrode to H 2 O 2 concentration is shown in Fig. 7. The inset plot shows the response current of the successive addition of 0.3 mM H 2 O 2 . From Fig. 6, it can be observed that the linear range is up to 12 mM with a correlation coefficient (R) of 0.9932 and then a plateau is reached gradually at higher H 2 O 2 concentrations. The biosensor has a good detection limit of 0.014 mM (signal-tonoise = 3), a high sensitivity of 22.42 nA/mM and a short response time (within ∼4 s). The apparent Michaelis-Menten constant (K app m ), which gives an indication of the enzyme-substrate kinetics, can be calculated from the electrochemical ver-sion of the Lineweaver-Burk equation [35,36]: Where I ss is the steady-state current after the addition of substrate, c is the bulk concentration of the substrate and I max is the maximum current measured under saturated substrate condition. K app m was determined by analysis of the slope and intercept for the plot of the reciprocals of the cathodic current versus H 2 O 2 concentration. The apparent Michaelis-Menten constant K app m in the present study is calculated to be 1.4 mM. This value is much smaller than reported in earlier work [34], indicating that the present electrode exhibits a higher affinity for H 2 O 2 . In Table 1 the analytical performance of this biosensor is compared with that of other biosensors in prior studies. The analytical characteristics of the enzyme electrode indicate that the immobilization of HRP mentioned above appears to be beneficial to the enhancement of the biosensor performance.

Reproducibility, selectivity and recovery studies
The reproducibility of the developed biosensor has To demonstrate the analytical applicability of the biosensors, the recoveries of four H 2 O 2 samples have been determined by the standard adding method. The results are satisfactory. As listed in Table 2, the recovery rate is in the range 95-101%. Table 2 Recovery studies of biosensor for determining H2O2.

Conclusions
In this investigation, we have fabricated a new hydrogen peroxide biosensor based on the immobilization of HRP into the NiFe 2 O 4 /chitosan nanocomposite. The experimental results clearly demonstrate that the immobilized HRP possesses excellent catalytic ability and well-retained activity. The developed biosensor is characteristic by its outstanding sensing performance, such as rapid response, wide linear range, and high sensitivity under optimum conditions.