Fabrication of AucoreCo3O4shell/PAA/HRP Composite Film for Direct Electrochemistry and Hydrogen Peroxide Sensor Applications

A completely new biosensor composed of cube-shaped AucoreCo3O4shell nanoparticles (AucoreCo3O4shell), polyacrylic acid (PAA), and horseradish peroxidase (HRP) modifi ed fi lm electrode was fabricated for the fi rst time. The biocompatibility and electrochemical properties of the resulting AucoreCo3O4shell-PAA-HRP composite film were studied by electrochemical impedance spectroscopy, UV-visible spectroscopy, and cyclic voltammetry. The UV-vis spectrum obtained suggests that HRP retains its native conformation in the modifi ed fi lm. The immobilized HRP shows a pair of quasireversible redox peaks at −0.31 V in 20 mM PBS (pH 7.0), and the biosensor shows a fast amperometric response to hydrogen peroxide with a linear range of 2.0×10−6 to 3.7×10−4 M. The kinetic parameters such as ks (electron transfer rate constant) and KM (Michaelis-Menten constant) are evaluated to be about 7.4 s−1 and 0.91 mM, respectively. These indicate that the cube-shaped AucoreCo3O4shell nanoparticles are an ideal candidate material for direct electrochemistry of redox proteins and for the construction of related enzyme biosensors, and that they may fi nd potential applications to biomedical, food, and environmental analyses and detection.


Introduction
Recently, nanomaterial-constructed biosensors have been attracting extensive interest because nanomaterials not only effectively retain the bioactivity of enzymes trapped in the biosensor but greatly facilitate the direct electron transfer between the enzyme and the electrode. (1)Excellent biocompatibility, stability, and sensitivity are crucial for biosensors to offer effective electron transfer channels between the redox-active enzyme and the electrode. (2)(5)(6)(7) For example, Dai et al. reported that a biosensor fabricated using tetragonal pyramid-shaped porous ZnO nanostructures has better biosensing properties than that fabricated using solid spherical ZnO nanoparticles. (4)Among nanomaterials, composite nanostructures usually exhibit better electrochemical properties than nanostructures possessing a single component because one dopant component amplifi es the electrochemical properties of the other component. (8,9)Ivnitski et al. integrated biologically derived silica with single-walled carbon nanotubes to fabricate a biosensing platform with excellent film-forming ability, good adhesion, biocompatibility, and bioelectrocatalytic properties. (5)obalt tetroxide (Co 3 O 4 ) is a spinel-type compound of 3d transition-metal oxides, (10) whose nanostructures show unique electronic and chemical properties and are widely applied in electrochemistry, (11) magnet, (12) and catalysis areas. (13)Currently, there is a broad research interest in the preparation of Co 3 O 4 with novel shapes and composite structures, such as nanotubes, (14) nanoboxes, (15) nanocubes, (16) nanohydrotalcites, (17) Co 3 O 4 / Fe 2 O 3 , (18) Co 3 O 4 /Si, (19) and Co 3 O 4 /ZnO. (20)We have synthesized Au core Co 3 O 4shell composite nanocubes, and demonstrated that they have better electrochemical performance than pure Co 3 O 4 nanocubes. (9)However, to the best of our knowledge, newly shaped or composite Co 3 O 4 nanomaterials are rarely used to construct biosensors, although biosensors based on Co 3 O 4 nanoparticles have been fabricated.For example, layered Co 3 O 4 nanofl akes with spongy nanostructure have been utilized to fabricate a hydrogen peroxide biosensor with high bioelectrocatalytic activity. (21)any researchers have been attempting to introduce inorganic nanomaterials and biomaterials into organic polymer matrices because forming ordered structures can better retain the bioactivity of biomaterials and enhance the properties of the composite materials. (22,23)(26) Among organic polymers, polyacrylic acid (PAA) as a water-soluble biocompatible polymer has good dispersing ability for and biocompatibility with watersoluble nanomaterials and enzymes, which has been used in biosensor fabrication and direct electrochemical studies. (27,28)n this study, we report a completely new biosensor composed of Au core Co 3 O 4shell nanocubes (Au core Co 3 O 4shell ), PAA, and a horseradish peroxidase (HRP)-modifi ed fi lm electrode.The biocompatibility and electrochemical properties of the Au core Co 3 O 4shell -PAA-HRP composite fi lm were studied by electrochemical impedance spectroscopy (EIS), UV-visible spectroscopy (UV-vis), and cyclic voltammetry (CV).

Methods: construction of the biosensor
A 3-mm-diameter glassy carbon (GC) electrode was fi rst polished with alumina (Al 2 O 3 ) slurry of successively smaller particles (1.0, 0.3, and 0.05 μm diameters).Then, the electrode was cleaned by ultrasonication in ultrapure water and ethanol, successively.In a typical procedure for the preparation of the PAA-Au core Co 3 O 4shell -HRP modifi ed electrode, the Au core Co 3 O 4shell colloid prepared as previously described (9) was firstly sonicated for 1 h to aid in the dissolution of the Au core Co 3 O 4shell , then equal volumes of pure PAA, 3.75 mg/ml HRP, and Au core Co 3 O 4shell colloidal solution were mixed.Next, 5 μl of the solution was cast onto the GC electrode surface with a 10-μl syringe.Finally, the modifi ed electrode was left to dry for over 20 h at room temperature.The modifi ed electrode was stored at 4°C in a refrigerator when not in use.
For comparison with the PAA-Au core Co 3 O 4shell -HRP modified electrode, a PAA/ Au core Co 3 O 4shell modifi ed electrode was also prepared using the same procedure described above.

Apparatus and measurements
All electrochemical measurements were performed at room temperature using a CHI 660C electrochemical workstation (CH Instru.Co., Shanghai, China).The measurements were based on a three-electrode system with the as-prepared fi lm electrodes as the working electrodes, a Ag/AgCl (3M KCl) electrode as the reference electrode, and a platinum wire as the auxiliary electrode.A 20 mM pH 7.0 PBS was used as the electrolyte in all the experiments.The buffer solution was deoxygenated by continuous sparging with highly purifi ed nitrogen for at least 30 min before the measurements, and a nitrogen atmosphere environment was maintained in all electrochemical measurements.
UV-visible absorption spectroscopy was carried out with a U-3010 spectrophotometer (Hitachi, Japan) using two 1-cm quartz cells.Sample fi lms for the measurement were prepared by casting PAA, Au core Co 3 O 4shell colloid, PAA-HRP, and PAA-Au core Co 3 O 4shell -HRP solutions onto quartz glass slides and drying them in air.The thus-obtained dry glass fi lms were examined.

Biocompatibility and electron transfer property characterization of the PAA-Au core Co 3 O 4shell -HRP composite fi lm
(31) Previous studies have proved that the biological activity of heme proteins depends on the Soret absorption band position of heme because it can provide information on the possible denaturation of heme proteins. (32,33)Figure 1 shows the UV-visible absorption spectra of HRP solution and the dry PAA, Au core Co 3 O 4shell , PAA-HRP, and PAA-Au core Co 3 O 4shell -HRP fi lms.No UV-visible absorption peaks of the dry PAA or Au core Co 3 O 4shell fi lm can be observed (curves a and b, respectively).However, characteristic Soret absorption bands at approximately 403 nm can be clearly observed in the UV-visible spectra of the dry HRP-PAA (curve d) and HRP-PAA-Au core Co 3 O 4shell (curve e) composite fi lms, which are approximately consistent with that of native HRP in pH 7.0 PBS (curve c).These results suggest that PAA and Au core Co 3 O 4shell could not lower the bioactivity of HRP or HRP trapped in a composite fi lm owing to these compounds having a similar structure to native-state HRP in PBS.EIS is a powerful tool for determining the interface properties of surface-modifi ed electrodes. (34)Figure 2 shows the electrochemical impedance spectra (Nyquist plots) of different modifi ed GC electrodes in 0.1 M pH 7.0 KCl solution containing 5 mM [Fe(CN) 6 ] 3−/4− (1:1).In the Nyquist plot of EIS, the semicircle portion at higher frequencies corresponds to an electron-transfer-limited process, and the linear portion at lower frequencies corresponds to a diffusion-controlled process. (35)In the bare GC electrode (curve b), electron transfer resistance (R ct ) is about 410 Ω.An obvious decrease in R ct (about 292 Ω) was observed when PAA was immobilized on GC electrode (curve a), indicating that the PAA may pro mote the electron transfer from the redox probe [Fe(CN) 6 ] 3−/4− to the underlying electrode.A marked increase in R ct (about 3000 Ω, curve d) was observed when HRP was immobilized on a GC electrode by cross-linking with PAA, which can be ascribed to the resistance of the macromolecular structure of HRP to electron transfer, and also confi rms the successful immobilization of HRP. (36)However, the R ct (about 2400 Ω) at the PAA-Au core Co 3 O 4shell -HRP/GC electrode decreases again (curve c), indicating that Au core Co 3 O 4shell nanocubes could facilitate electron transfer.and the underlying electrode.(39) The peak-to-peak separation ΔEp, which is directly related to electron transfer rate, is about 50 mV.Such a small ΔEp reveals a fast and quasi-reversible electron transfer process.Figure 4 shows typical cyclic voltammograms of the PAA-Au core Co 3 O 4shell -HRP/GC electrode with scan rates from 0.05 to 0.80 V/s.As studied previously, (40) besides an increasing peak separation, both cathodic and anodic peak currents of HRP increase linearly with increasing scan rate, as shown in the inset of Fig. 4; moreover, ΔEp increases with scan rate, indicating that all the electroactive HRP in the fi lm was reduced on the forward cathodic scan and the reduced HRP was then converted to the oxidized form on the reverse anodic scan.This result indicated that the PAA-Au core Co 3 O 4shell composite fi lm provides a friendly microenvironment for the captured HRP to enable the electron transfer between HRP and the GC electrode and it is a surface-confi ned electrochemical process.According to Faraday's law (Q = nFAΓ*), the average surface concentration of electroactive HRP (Γ*) in the PAA-HRP fi lm is about 6.80×10 −11 mol/cm 2 (assuming a one-electron transfer reaction), which is about 2 times higher than that of HRP in chitosan and approximately 3 times higher than that of the theoretical monolayer coverage. (41)This result indicates that multilayers of HRP entrapped in the as-prepared fi lm share in the electron-transfer process.The kinetic parameter k s was estimated using the model of Laviron: (42)

Direct electrochemistry of the immobilized HRP
where α is the charge transfer coeffi cient, k s is the heterogeneous electron transfer rate constant, ΔEp is the peak separation, n is the number of electrons transferred in the rate-determining reaction, R is the gas constant, T is the absolute temperature, and υ is the scan rate.According to the method of Laviron, taking charge transfer α to be 0.5 and scan rate to be 0.8 V/s with ΔEp 75 mV, the k s of HRP is calculated to be 7.4 s −1 , suggesting a fast electron transfer process.

Electrocatalytic reduction of H 2 O 2
HRP immobilized on an electrode surface usually has excellent electrocatalytic activity towards H 2 O 2 . (24)Figure 5 shows the electrocatalytic properties of H 2 O 2 solutions of different concentrations at the PAA-Au core Co 3 O 4shell -HRP/GC electrode.The cathodic peak current at approximately −0.35 V (vs Ag/AgCl) apparently increases, whereas its corresponding anodic peak current decreases as H 2 O 2 is successively added to PBS, suggesting a representative electrocatalytic reduction of H 2 O 2 .Moreover, the cathodic peak current increases linearly within a certain range of H 2 O 2 concentrations (inset of electrocatalytic reduction of H 2 O 2 at the HRP-based enzyme electrode should be similar to that reported recently, which can be expressed as follows: (43) HRP[Heme(Fe The overall reaction of ( 1)-( 5) would be Figure 6 shows a plot of the variation in electrocatalytic current (I cat ) against H 2 O 2 concentration for the PAA-Au core Co 3 O 4shell -HRP/GC electrode in 20 mM pH 7.0 PBS.Along with increasing H 2 O 2 concentration from 2 to 370 μM, I cat linearly increases, indicating that the linear range is from 2.0×10 −6 to 3. when the signal-to-noise ratio (S/N) is 3.However, when higher H 2 O 2 concentrations were added, the plot tends to level off, showing a typical Michaelis-Menton process.
According to the Lineweaver-Burk equation, (44) where I ss is the catalytic current, c is the bulk concentration of the substrate, I max is the maximum catalytic current, and K M is the Michaelis constant.Herein, the Lineweaver-Burk plot gives a K M of 0.91 mM for the PAA-Au core Co 3 O 4shell -HRP electrode (inset in Fig. 6).
The small K M indicates that the PAA-Au core Co 3 O 4shell -HRP electrode has a high catalytic effi ciency for the reduction of H 2 O 2 over a wide linear range.
To further study the performance of the biosensor, the amperometric response of the as-prepared biosensor for H 2 O 2 was also investigated by amperometric current-time curve method.On the basis of the optimal experiments, a constant potential of −0.35 V was selected as the applied potential for high sensitivity.Figure 7 shows a typical current-time curve of the biosensor (PAA-Au core Co 3 O 4shell -HRP/GC electrode) for 6, 10, and 20 μM H 2 O 2 solutions added successively to pH 7.0 PBS.With a stepwise increase in H 2 O 2 concentration in the stirred PBS, the biosensor responded rapidly to the substrate and a stepwise growth in reduction current was observed.The response time of about 2 s (achieving 95% of the steady-state current) indicates a fast process and the immobilized HRP could catalyze H 2 O 2 well.Such a fast response can be ascribed to the fast diffusion of the substrate in the composite fi lm.

Stability and reproducibility of the PAA-Au core Co 3 O 4shell -HRP composite fi lm
To widen the range of biosensor applications, besides high sensitivity and good biological activity, reproducibility and stability are of key importance for a biosensor.When the PAA-Au core Co 3 O 4shell -HRP/GC electrode was used to successively scan 15 circles at a scan rate of 0.20 V/s, no obvious changes appear in the cyclic voltammograms.The relative standard deviation (R.S.D.) of the biosensor produced is 0.91% for 15 successive measurements, suggesting that the biosensor has excellent reproducibility.
HRP biosensor stability can be evaluated by measuring the cyclic voltammetric peak current of HRP at long-term time intervals.The biosensor was stored under dry condition at 4°C when it was not in use.The decrease in cathodic peak current is less than 2.0% after 40 days, indicating that the as-prepared biosensor has good long-term stability.This stability could be attributed to the excellent biocompatibility and favorable microenvironment of the sensor for HRP to retain its bioactivity.

Conclusions
In summary, we have designed a novel biosensor composed of PAA, HRP, and Au core Co 3 O 4shell composite film.The method we used may be extendable to the preparation of other biosensors constructed with polymers, nanomaterials, and redoxprotein molecules.Our results demonstrate that HRP effectively retains its native structure in PAA-Au core Co 3 O 4shell -HRP composite films, at which direct electron transfer and electrocatalytic activity could be effectively achieved.Also, the asprepared biosensor was demonstrated to possess multilayer coverage and exhibit good reproducibility, stability, fast response, and excellent biological activity.For example, the biosensor has a fast amperometric response (about 2 s) to H 2 O 2 with a linear range of 2.0×10 −6 to 3.7×10 −4 M, and has a k s and a K M of about 7.4 s −1 and 0.91 mM, respectively.The unique polymer-nanomaterial composite materials used are expected to offer a good biosensing platform for other redox proteins and to fi nd applications in biosensing, biocatalysis, and biodetection.

Figure 3 Fig. 2 .
Figure3shows representative cyclic voltammograms of the PAA-Au core Co 3 O 4shell / GC and PAA-Au core Co 3 O 4shell -HRP/GC electrodes in 20 mM PBS at a scan rate of 0.20 V/s.At the PAA-Au core Co 3 O 4shell /GC electrode, no redox peak was observed (curve a), indicating that both PAA and Au core Co 3 O 4shell do not undergo electrochemical reactions in the potential range studied.However, stable, nearly reversible, and well-defi ned peaks for HRP-Fe III /Fe II redox couple were observed at the PAA-Au core Co 3 O 4shell -HRP/GC electrode (curve b), which could be ascribed to the direct electron transfer between HRP

Fig. 5 .
Figure 6 shows a plot of the variation in electrocatalytic current (I cat ) against H 2 O 2 concentration for the PAA-Au core Co 3 O 4shell -HRP/GC electrode in 20 mM pH 7.0 PBS.Along with increasing H 2 O 2 concentration from 2 to 370 μM, I cat linearly increases, indicating that the linear range is from 2.0×10 −6 to 3.7×10 −4 M (R = 0.9991, n = 10).The detection limit of H 2 O 2 at the PAA-Au core Co 3 O 4shell -HRP electrode is about 9.0×10 −7 M