Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe3O4 Magnetic Nanoparticle Bio-Complex Film

A DNA/chitosan-Fe3O4 magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe3O4 nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe3O4/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe3O4 and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe3O4 film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s−1. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe(III)) and HRP(Fe(II)). The obtained electrode also displayed an electrocatalytic reduction behavior towards H2O2. The resulting DNA/chitosan/Fe3O4/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm−2·M−1) toward H2O2. A linear response to H2O2 measurement was obtained over the range from 2 μM to 100 μM (R2 = 0.99) and an amperometric detection limit of 1 μM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.


Introduction
The direct electron transfer (DET) between electrodes and redox proteins, particularly enzymes, has stimulated increasing interest because of its significance in both theoretical and practical applications in electrochemistry, such as fabricating biosensors, enzymatic bioreactors, and biomedical devices [1][2][3]. Nevertheless, DET between the enzyme and a conventional electrode is usually prohibited because enzymatic redox centers are deeply embedded in the structure of the enzyme. Therefore, new materials, such as carbon nanotube [4][5][6], quantum dot [7], nanoparticles [8], graphene [4], hybrid organic-inorganic film [9], room temperature ionic liquid matrix [10] and mesoporous matrix [11] are required to establish an electrical connection between these redox centers and the electrode for fabricating a third-generation enzyme biosensor or a mediatorless enzymatic biofuel cell.
In recent years, there has been an increasing trend in the design and development of magnetic nanoparticles for bioanalytical applications [12,13]. Magnetic nanoparticles have been considered as an interesting material for the immobilization of desired biomolecules because of some superior properties: electro-conductivity, bio-compatibility and ease of synthesis [14]. Enzyme-immobilized magnetic nanoparticles could potentially lead to unique properties such as large surface area, high bioactivity, and excellent stability [15,16]. However, pure iron oxide nanoparticles may not be very useful in biomedical and technological applications because they are very likely to aggregate, and they have limited functional groups for selective binding [17]. As a result, a number of biomaterial-functionalized magnetic nanoparticles have been used in bioelectronic applications [12][13][14][15][16][17], such as poly(ethyleneimine) layer-functionalized magnetic nanoparticles [18], Au-polydopamine-Fe 3 O 4 magnetic nanoparticles [19], and magnetic core-shell Fe 3 O 4 @Al 2 O 3 nanoparticles [20], which were used for capture of heme proteins for direct electrochemistry. Unfortunately, these methods may involve complicated electrode modification procedure or costly modification regents.
DNA/polycation based biocompatible films are formed by natural DNA molecules (negatively charged polyanions) and natural polycations molecules based on the electrostatic force of attraction. As novel electrochemical recognition layers, DNA/polycation based biocompatible films possess a number of unique properties [21][22][23]: (1) biocompatible microenvironment around the enzyme; (2) a host matrix of electrochemically active species (e.g., redox active intercalators) and metal ions which specifically binds to double-stranded DNA; (3) unique electron transfer property improving electron transfer characteristics between redox active species and the electrode surface; and (4) simplicity in procedure. In our previous reports, DNA/poly(allylamine) films have been successfully used as the support matrixes for co-immobilization of electron mediator-methylene blue and HRP [22][23][24], and immobilization of electrocatalytic element-copper ion [23,25] to fabricate novel amperomeric biosensors. More recently, glucose oxidase (GOD) was effectively immobilized on a DNA/chitsoan films modified glassy carbon electrode (GCE), and the direct electrochemistry of GOD and biosensing for glucose were performed successfully [26].
The DET of immobilized heme proteins such as horseradish peroxidase (HRP) [3], cytochrome C [27], and hemoglobin [28] is based on the Fe (III) /Fe (II) conversion in the active heme center of the proteins. Among them, HRP has been widely used for the fabrication of amperometric biosensors based on its direct electrochemistry to detect H 2 O 2 due to its high purity, sensitivity, low cost and availability [29]. There are many ways to achieve the DET of immobilized HRP, for instance, using sol-gel matrices [30], biopolymers [31], and nanomaterials [32].
In this paper, a new type of DNA/chitosan/Fe 3 O 4 magnetic nanoparticle bio-complex film was constructed for the immobilization of HRP. DNA/chitsoan film and Fe 3 O 4 nanoparticles with the excellent biocompatibility and good conductivity were used to maintain the native structure of HRP and to facilitate the direct electrochemistry of HRP in the biofilm. Although a Fe 3 O 4 /chitosan/HRP-modified glassy carbon electrode has been reported for amperometric detection of H 2 O 2 [33], methylene blue in solution was needed as an electron transfer mediator to transfer the electron between the HRP and electrode in the system. Here, the direct electron transfer and electrocatalysis of HRP based on the DNA/chitosan/Fe 3 O 4 film was studied.

Morphologies of DNA/Chitosan/Fe 3 O 4 /HRP Film Surface
The surface morphologies of

Electrochemical Impedance Spectroscopy (EIS) of DNA/Chitosan/Fe 3 O 4 /HRP Film
EIS can provide useful information on the impedance changes of the electrode surface during the fabrication process. In EIS, the diameter of a semicircle in the high frequency region corresponds to the electron transfer resistance, R et . This resistance controls the electron transfer kinetics of redox probe at the electrode interface. Figure    The formal potential (E 0 ′) was estimated to be ~ −0.32 V (versus Ag/ACl in saturated KCl) by the average of the cathodic and anodic peak potentials. Therefore, it can be concluded that the redox waves should be ascribed only to HRP, which is characteristic of quasi-reversible DET process of HRP[Fe (III) ] and HRP[Fe (II) ] in the HRP previously reported in various films [30][31][32]. Thus, DET of HRP in the DNA/chitosan/Fe 3 O 4 film has been achieved successfully. Similar to the case of DNA/chitosan/Fe 3 O 4 /HRP/GCE (curve c), the DNA/chitosan/HRP modified electrode (curve d) showed a pair of quasi-reversible redox peaks, but the oxidation and reduction peak currents were 87% and 53%, respectively, smaller than the corresponding peak currents obtained at DNA/chitosan/Fe 3 O 4 /GCE. This result clearly indicated that the electron transfer of HRP on the DNA/chitosan electrode was amplified by incorporation of Fe 3 O 4 magnetic nanoparticles.  Figure 4). The linear relationship between peak current and scan rate indicated that the redox process of HRP in the film was a surface-confined process. The electron transfer rate constant (k s ) has been estimated from the peak potential separation value using the model of Laviron [34]. Taking a charge transfer coefficient α of 0.5, the electron transfer rate constant of HRP at the DNA/chitosan/Fe 3 O 4 /HRP/GCE was 3.7 s −1 . The electron transfer rate is higher than the values reported for HRP immobilized in a polystyrene and multiwalled carbon nanotube composite film (1.15 s −1 ) [32], a colloidal gold modified screen-printed electrode (0.75 s −1 ) [35], and a dipalmitoyl phosphatidic acid film (1.13 s −1 ) [36]. Since DNA/chitsoan film and Fe 3 O 4 nanoparticles with the excellent biocompatibility and good conductivity appeared to be capable of maintaining the native structure of HRP, it suggested that DNA/chitosan/Fe 3 O 4 film was an excellent promoter for the direct electron transfer between HRP and GCE. The DET of HRP immobilized on the DNA/chitosan/Fe 3 O 4 /HRP/GCE showed a strong dependence on solution pH. Figure 5 shows the CVs of the DNA/chitosan/Fe 3 O 4 /HRP/GCE in phosphate buffer at different pH values. CVs with stable and well defined peaks were observed in the pH range 5.0-8.0, but increasing pH caused a negative shift of both cathodic and anodic peak potentials. This is attributed to the involvement of proton transfer in the HRP[Fe (III) ]/HRP[Fe (II) ] redox couple. The E 0 ′ value of HRP varied linearly in the range of pH from 5.0 to 8.0, with a slope of 56.14 mV pH −1 (inset graph in Figure 5). This value is very close to the theoretical value for the transfer of one proton and one electron in a reversible reduction (58 mV pH −1 at 25 °C) [37].

Electrocatalysis of DNA/Chitosan/Fe 3 O 4 /HRP/GCE
By using hydrogen peroxide as a probe, the electrocatalytic properties of the HRP in DNA/chitosan/Fe 3 O 4 /GCE were studied. Figure 6 shows the bioelectrocatalytic activity of the HRP in DNA/chitosan/Fe 3 O 4 /GCE toward the reduction of H 2 O 2 at a scan rate of 0.1 V· s −1 . A pair of quasi-reversible CV peaks appeared in the absence of H 2 O 2 (curve a). Upon the addition of H 2 O 2 to the pH 7.0 phosphate buffer, the reduction peak current of the immobilized HRP increased dramatically and the oxidation peak current decreased concomitantly (curve b, c and d). The reduction peak current increases with increasing concentration of H 2 O 2 (curve b, c and d), indicating that an electrocatalytic reduction of H 2 O 2 took place at electrode and the HRP entrapped in the DNA/chitosan/Fe 3 O 4 /HRP film maintained its bio-electrocatalytic activity. Similar electrocatalytic behavior has been reported at Nafion/HRP/graphene/GCE [3] and HRP-incorporated in dipalmitoyl phosphatidic acid film-modified pyrolytic graphite electrode [36].
The electrocatalytic mechanism of HRP immobilized in the DNA/chitosan/  (2) and (3) [39].       According to the intercept and slope of above regression equation, K m was estimated to be 0.28 mM. The value is much smaller than 0.66 mM for HRP immobilized in a polystyrene and multiwalled carbon nanotube composite film [32], and 0.818 mM for HRP immobilized in γ-Al 2 O 3 nanoparticles/chitosan film [40].   Figure 8.
The long-term stability of electrode was investigated by examining its current response after storage in a refrigerator at 4 °C. The electrode exhibited no obvious decrease in current response in the first week and maintained about 95% of its initial value after three weeks. The relative standard deviation (R.S.D.) of the electrode response to 10 mM H 2 O 2 for 5 successive measurements was 2.1%, indicating an acceptable electrode reproducibility. The selectivity of electrode was performed by comparing the amperometric response of 0.2 mM H 2 O 2 before and after adding 2 mM of several known interfering species, respectively, in 0.1 M phosphate buffer (pH 7.0). The steady-state amperometric current ratio obtained in the presence to that in the absence of each of these interfering species is 0.98 for glucose, 0.93 for ascorbic acid and 0.96 for uric acid. Notably, there was minimal interference from glucose, ascorbic acid and uric acid in the determination of H 2 O 2 . The good selectivity of this electrode is largely attributed to the low working potential (−0.25 V).

Preparation of DNA/Chitosan/Fe 3 O 4 /HRP/GCE
A GCE [3 mm diameter, Bioanalytical Systems (BAS), West Lafayette, IN, USA] was polished with 0.05 µm alumina slurry, rinsed with water, sonicated in water for 2 min, and dried. The DNA/chitosan/Fe 3 O 4 /HRP/GCE was prepared as follows: aqueous solutions of chitosan (0.5 mL, 1 mg/mL) and Fe 3 O 4 (0.5 mL, 1 mg/mL) were mixed for 15 min. Then, 10 µL of dsDNA (1 mg/mL), 20 µL of the mixture of chitosan and Fe 3 O 4 , and 10 µL of HRP (1 mg/mL) aqueous solution were successively placed on the GCE surface to form a polyion complex layer. The electrode was allowed to dry for 24 h under a 1000 mL beaker at room temperature. After being rinsed with distilled water, the resulting DNA/chitosan/Fe 3 O 4 /HRP/GCE was stored in the refrigerator at 4 °C when not in use. Before electrochemical measurements, the electrodes were immersed in phosphate buffer for 15 min.

Electrochemical Measurements
All electrochemical experiments were performed with a conventional three-electrode system using a CHI 660D electrochemical workstation (Shanghai CH Instruments, Shanghai, China). An Ag/AgCl (sat. KCl) electrode, a platinum wire electrode (1 mm diameter) and a GCE were used as the reference electrode, the counter electrode, and the working electrode, respectively. CV and constant potential amperometry were carried out by using a deoxygenated (N 2 -saturated) 0.1 M phosphate buffer (10 mL). Deoxygenated electrolyte solutions were prepared by bubbling high purity grade nitrogen gas through the solution at least 20 min prior to the electrochemical measurements. EIS measurements were performed with 0.1 M KCl solution containing 5 mM [Fe(CN) 6 ] 3−/4− under applied potential of 170 mV with the frequency range from 0.01 to 100,000 Hz with the amplitude of 8 mV. All measurements were done at room temperature.

Scanning Electron Microscopic Analysis
The SEM analysis of DNA/chitosan/Fe 3 O 4 /HRP polyion complex membrane was performed using a JEOL-6480LV microscope (JEOL Ltd., Tokyo, Japan) operating at 15.0 kV. Prior to SEM analysis, ca. 10 nm of carbon film was sputtered on the samples.

Conclusions
As one kind of novel nanomaterial, magnetic nanomaterials not only have the common nano-characters, but also have some special properties. Acting as the carrier of biological molecules, DNA/chitosan/Fe 3 O 4 film can provide a microenvironment which is similar to biological molecules of internal environment. In this paper, a simple method for constructing HRP modified electrode on a DNA/chitosan/Fe 3 O 4 bio-magnetic polyion complex membrane to realize direct electron transfer and electrocatalysis was proposed. The results show that HRP was successfully immobilized on the electrode surface by DNA/chitosan/Fe 3 O 4 bio-polyion complex membrane. The HRP on the electrode exhibited fast electron transfer rate, high affinity to H 2 O 2 and good bioactivity toward H 2 O 2 reduction.