Peroxidase mimic activity of hematite iron oxides (α-Fe₂O₃) with different nanostructures

Abstract

Enzyme mimics have garnered considerable attention as they can overcome some serious disadvantages associated with the natural enzymes. In recently developed sphere and rod shaped iron oxide peroxidase mimic nanoparticles, the influence of physical parameters such as shape, size and surface area on the catalytic performance was not clearly demonstrated. In order to better understand the influence of physical parameters on the enzyme mimic activity of iron oxide nanoparticles, the present study was initiated using three different shaped hematiteα-Fe₂O₃ nanostructures, particularly hexagonal prism, cube-like and rods as model systems. A comparative account of kinetic parameters (K_m, V_max and K_cat) of the peroxidase mimic activity by the various α-Fe₂O₃ nanostructures indicated that the enzymatic potential of these nanoparticles increased from hexagonal prism to rods, via cube-like, suggesting that one-dimensional particles act as a more efficient enzyme mimic system compared to their multi-dimensional counterparts. Surface area is likely to be a key physical aspect responsible for the enzyme mimic activity. Interestingly, however, particles with lower surface area showed better catalytic performance in the case of one-dimensional rod structure. Upon further analysis of the one-dimensional rods, additional physical properties such as porosity and pore shape also seem to have a significant contribution to their catalytic activity.

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1. Introduction

Chemical or artificial synthesis of enzymes is a rapidly developing field due to the drawbacks associated with the natural enzymes such as denaturation by proteases, requirement of special storage conditions and the cost factor involved. Some level of success has been achieved in this direction with the development of chemically synthesized alternatives for serine proteases,¹hematin,² and superoxide dismutase.³ In addition, a recent finding demonstarted that iron oxide nanoparticles have the ability to catalyze oxidation reactions, similar to that of natural peroxidase enzyme,⁴ giving rise to new possibilities in the field of enzyme mimics or artificial enzymes.

Shape has been recognized as an important parameter influencing nanoparticle properties. Particularly, one-dimensional (1D) structures such as nanorods and nanowires possess unique properties that are different from their bulk materials and even the corresponding zero-dimensional counterparts (spherical nanocrystals).^5,6 This was also seen in the recently developed peroxidase mimic nanoparticles where the peroxidase mimic activity of rod type Fe₃O₄ dominated over the spheres which were much smaller in size.⁷ It raised an interesting question over the involvement of various physical parameters like shape, size, surface area and dimensionality in the enzyme mimic activity of nanoparticles. This inspired us to carry out a systematic study that may help in identifying a possible correlation between peroxidase mimic activity and the physical properties of iron oxide nanoparticles. In order to do so, stable hematiteα-Fe₂O₃ nanoparticles of various shapes (hexagonal prism, cube-like and rod) were chosen as model systems.

Hematite (α-Fe₂O₃), an n-type semiconductor with 2.1 eV band gap, is the most stable, non-toxic, corrosion-resistant iron oxide under ambient conditions. Due to its low cost, high resistance to corrosion and being environmentally safe, this metal oxide has been used in cellular delivery,⁸ imaging,⁹cell separation,¹⁰ biosensing¹¹ and for evaluation of cellular response towards the nanomaterials.¹²Hematiteα-Fe₂O₃ nanoparticles used in this work are different from previously used Fe₃O₄ nanoparticles in terms of chemical composition, structure, size and synthesis route. Although both Fe₃O₄ and α-Fe₂O₃ are ferromagnetic in nature, they differ on the basis of net magnetic moment and magnetic crystal structure.^13,14α-Fe₂O₃ nanoparticles of varying shape (hexagonal prisms, cube-like and rods) used for this study are synthesized through simple, aqueous and surfactant-free routes.^15,16 Such synthesis procedures utilizing green approaches^17–19 yield biocompatible iron oxides with much lesser toxicity,²⁰ which facilitates their direct use for biological and biochemical application. In this study we analyzed the influence of various physical parameters like shape, size, surface area and dimensionality on the enzyme mimic properties of α-Fe₂O₃ nanomaterial. Kinetic parameters (V_max and K_m) of all the nanoparticles were assessed, and a correlative investigation between the various physical features of α-Fe₂O₃ and its enzyme mimicking property is presented. It was shown for the first time that α-Fe₂O₃ nanoparticles possess peroxidase-like activity and also serve as model systems to give understanding of the influence of various physical parameters on the catalytic activity of the nanoparticles.

2. Experimental section

2.1. Synthesis of α-Fe₂O₃ nanostructures

2.1.1. Materials and methods. All chemicals used for the synthesis of nanomaterials and enzyme kinetic assay such as iron chloride (FeCl₃·6H₂O), urea ((NH₂)₂CO), caffeine, citric acid, sodium hydrogen phosphate (Na₂HPO₄) and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich and used without any further purification. A stable, chromogenic and non-carcinogenic horse radish peroxidase (HRP) substrate, TMB (3,3′,5,5′-tetramethyl benzidene), was purchased from Sigma-Aldrich, and hydrogen peroxide (H₂O₂) from Samchun Pure Chemicals, South Korea.

2.1.2. Synthesis of α-Fe₂O₃ nanostructures. α-Fe₂O₃ nanoparticles with different structures (hexagonal prism, cube-like, and rods) were synthesized using aqueous routes. To begin with, α-Fe₂O₃ nanorods were synthesized through hydrolysis of iron chloride (FeCl₃·6H₂O) by a hydrothermal method with slight modification in the earlier synthesis route.¹⁵ In a typical synthesis procedure, 0.02 M iron chloride solution was prepared in preheated deionized water at 90 °C. After a certain period of time, varying concentrations of HCl were added, which is a key factor in controlling size, leading to the formation of different sized β-FeOOH nanorods. The respective flasks were magnetically stirred at 90 °C for 24 h, culminating into yellow coloured mixtures. Thus obtained yellow coloured mixtures are indicative of the formation of β-FeOOH nanorods and were cooled to room temperature, centrifuged, washed repeatedly with deionized water and ethanol and then dried overnight at 80 °C in an oven. The as-synthesized β-FeOOH nanorods were then used as a precursor for various uniform α-Fe₂O₃ nanorods. The β-FeOOH rods were slowly heated at the rate of 2 °C min⁻¹ upto 250 °C and maintained for 2 h under static conditions. This procedure transforms the precursors to brown coloured hematite α-Fe₂O₃ rods. During the transformation, apparent shape and size of the β-FeOOH were maintained in the resulting α-Fe₂O₃ nanorods. The resultant rods with different lengths were the outcome of different HCl concentrations used during the synthesis procedure and are labelled R-1, R-2, and R-3 according to their length. Based on our previous work, the α-Fe₂O₃ hexagonal prisms were synthesized using urea as a reducing agent.¹⁶ Briefly, a certain amount of urea was dissolved in preheated deionized water at 90 °C with vigorous stirring, followed by addition of iron chloride. The molar ratio of iron chloride to urea used was kept at 1.0 : 7.5. After addition of iron chloride, the colour of the solution changed to brown. The solution was stirred for 12 h at the same temperature and the resulting nanostructures were isolated by centrifugation with repeated washings.

In addition, cube-like α-Fe₂O₃ particles were synthesized using 0.06 mol of iron chloride (FeCl₃·6H₂O) and 0.03 mol of caffeine instead of hydrochloric acid or urea in aqueous solution. The flask was magnetically stirred for 24 h at 90 °C. The mixture was allowed to cool naturally to room temperature, and the resultant brown product, also indicative of formation of hematite, was isolated through centrifugation with successive washings by deionized water and ethanol and dried at 80 °C overnight.

2.2. Characterization of α-Fe₂O₃ nanostructures

The as-synthesized α-Fe₂O₃ nanostructures were characterized by using various analytical techniques. The microscopic features of the samples were elucidated with a transmission electron microscope (TEM, EM 912 Omega) operated at 120 kV and a scanning electron microscope (SEM) (SEM, LEO 1455VP, Hitachi S-4700) operated at an acceleration voltage of 25 kV. X-Ray diffraction (XRD) patterns of the samples were obtained with a Rigaku 1200 diffractometer with CuKα radiation using a Ni β-filter at a scan rate of 4° min⁻¹. The X-ray source was operated at 40 kV and 30 mA.

Biocatalytic and enzyme mimic activities of all iron oxide nanoparticles are considered to be mostly a surface governed phenomenon.⁴ Hence, surface area is an important aspect for analyzing the enzyme mimic activity of the biocatalytic nanomaterials. N₂ adsorption–desorption isotherms were conducted at 77 K on a KICT SPA-3000 gas adsorption analyzer after the test materials were degassed at 423 K to 20 μTorr for 12 h. Surface areas of the various materials were determined from the adsorption branch of nitrogen isotherms in a relative pressure range from 0.05 to 0.2 using the Brunauer–Emmett–Teller (BET) equation. Total pore volumes were determined from the amount of gas adsorbed at the relative pressure of 0.99. Any apparent physical changes in the α-Fe₂O₃ nanorods after the isotherm measurements were not detected and were confirmed on the basis of the microscopic analysis of the samples by SEM and TEM.

2.3. Peroxidase mimic activity of α-Fe₂O₃ nanostructure

2.3.1. Optimal concentration determination. The minimum α-Fe₂O₃ nanoparticle concentration which showed maximal peroxidase enzyme mimic activity was determined. Various concentrations of α-Fe₂O₃ were tested with increasing concentrations of the peroxidase substrate TMB in citrate buffer at pH 4, and finally 3.0 mM of α-Fe₂O₃ was chosen as an optimal working concentration, showing maximal peroxidase mimic activity for all the three different shaped nanoparticles, namely, hexagonal prism, cube-like, and rod nanoparticles.

2.3.2. Kinetic assay of α-Fe₂O₃ nanoparticles. It is well known that HRP catalyzes oxidation of its substrate, resulting in a colour change of the reaction. Such reactions can be normally quenched by addition of sulfuric acid, thus allowing for recording absorbance and further analysis. Peroxidase mimic activities of all the nanoparticles, namely, hexagonal prism, cube-like, and different rods were characterized by performing a steady state kinetic assay. TMB was used as a substrate. Clear and transparent stock solution was prepared by dissolving powdered TMB in DMSO. Serial dilutions of various TMB concentrations were done using citrate buffer of pH 4. For all the experiments, a fresh buffer was reconstituted from the component solutions. All the reactions were carried out in 1.5 ml amber coloured vials and incubated at 37 °C for 30 min. For steady state kinetic studies, 100 μl of respective α-Fe₂O₃ nanoparticles were added with equal amounts of solutions having increasing concentrations of TMB in citrate buffer. Reactions were initiated by addition of 30% H₂O₂. Whole solutions were mixed well in each vial and then subjected to incubation. All reactions were quenched by addition of 2.0 M sulfuric acid immediately after the incubations were over. Reaction vials were centrifuged for 30 s and 100 μl of the supernatant was dispensed in 96 well plates for recording the absorbance using a microtitre plate reader (Molecular Devices E_max USA) at 450 nm. All the experiments for each nanoparticle were repeated thrice for reproducibility. Appropriate controls were used wherever necessary in due course of the experimentation. The kinetic parameters were determined via Michaelis–Menten equation: v = V_max[S]/(K_m + [S]), where v is the initial velocity of the reaction, V_max is the maximal rate of reaction, [S] is the substrate concentration, and K_m is the Michaelis–Menten constant.

3. Results and discussion

3.1. Structural parameters of α-Fe₂O₃ nanoparticles

Hexagonal prisms have average parallel and diagonal sides measuring around 80–90 nm and 60–70 nm along with width and thickness ranging between 105–110 nm and 85–95 nm, respectively. Cube-like nanoparticles have an average size of 100 nm, whereas rod shaped particles have size varying between 250–400 nm in length and 30–35 nm in diameter. The N₂ adsorption–desorption isotherms for the hexagonal prism, cube-like, and rod shaped α-Fe₂O₃ are provided in the ESI† (Fig. S1–S3), and the surface area details are presented in Table 1. The XRD patterns of all the iron oxide nanostructures are shown in ESI† (Fig. S4 to S6). Despite variations in shape, all the α-Fe₂O₃ nanostructures reveal similar XRD patterns, confirming that all the as-synthesized iron oxide nanostructures correspond to the crystalline hematite α-Fe₂O₃ phase with lattice parameters a = 5.037 Å and c = 13.75 Å. These values are well in agreement with the literature values (JCPDS 89-0597) for hexagonal prism and cube-like nanoparticles along with the values (JCPDS 33-0664) for nanorods. All the reflection peaks are sharp and readily indexed to a pure rhombohedral phase of α-Fe₂O₃, suggesting that the particles are highly crystalline. It can be seen that these samples have a pure phase since no other peaks are observed for the presence of impurities. Length distribution was estimated by measuring lengths of particles from randomly selected regions of the respective TEM images. Distribution plots for R-1, R-2 and R-3 are provided in Fig S7, ESI.† Most of the particles were found to be of average length as mentioned in Table 1.

Table 1 Comparison of morphological features and enzyme mimic activity of various α-Fe₂O₃ nanoparticles with respect to their relative K_m, V_max and K_cat values

Morphology	Size/nm	Surface area/m^2 g^−1	K m/mM	V max/nM s^−1	K cat/s^−1
Hexagonal prism	110 ± 10	43 ± 4	0.768 ± 0.091	5 ± 0.44	1.66
Cube-like	100 ± 7	47 ± 5	0.957 ± 0.085	6.3 ± 0.35	2.2
Rod shaped (R-1)	31 ± 5 (diameter)	55 ± 5	0.257 ± 0.089	16.5 ± 2.1	5.5
	380 ± 20 (length)
Rod shaped (R-2)	30 ± 3 (diameter)	82 ± 8	0.317 ± 0.071	15.3 ± 1.81	5.1
	330 ± 20 (length)
Rod shaped (R-3)	30 ± 3 (diameter)	111 ± 10	0.518 ± 0.047	10.60 ± 1.38	3.53
	278 ± 20 (length)

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3.2. Peroxidase mimic activity analysis by steady state kinetic assay of α-Fe₂O₃ nanoparticles

The current study began with a correlative assessment of various physical parameters such as shape, size, and surface area, and their influence on peroxidase mimic activity of α-Fe₂O₃ nanoparticles. The enzyme like activity of α-Fe₂O₃ originates because of the presence of ferrous ions at the surface of the nanoparticles. The ferrous ions interact with the substrate in the presence of peroxide, resulting in a coloured reaction product. The mechanism most likely follows Fenton’s reaction²¹ and can be written as follows:

Fe²⁺ + H₂O₂ → Fe³⁺ + OH˙ + OH⁻ (1)

Fe³⁺ + H₂O₂ → Fe²⁺ + OOH˙ + H⁺ (2)

The hydroxyl radicals (OH˙) formed in step (1) catalyse the oxidation of the TMB substrate, resulting in a coloured product. All the three different shaped α-Fe₂O₃ nanoparticles chosen were found to catalyze the reaction with the peroxidase substrate TMB in the presence of H₂O₂, producing a blue coloured product (Fig. 1–1) having absorbance maxima at 652 nm. The reactions were stopped by addition of 2.0 M sulfuric acid and the resultant blue coloured product immediately changed to yellow (Fig. 1–2). The yellow coloured product so obtained was measured using a microtitre plate reader at the maximum absorbance of 450 nm. After recording the respective absorbances for all the nanoparticles, SigmaPlot®10.0 software was used to determine the V_max and K_m values. Michaelis–Menten plots (data not shown) and Lineweaver–Burk plots were drawn for all the nanomaterials. The catalytic constant K_cat was calculated using eqn (3):

K_cat = V_max/[E] (3)

where [E] represents the nanoparticle concentration.

Fig. 1 Peroxidase enzyme-like activity of α-Fe₂O₃ hexagonal prism (first row), cube-like (second row), and rod-shaped nanoparticles (third row), showing (1) blue colour development after 30 min, and (2) yellow colour development after stopping the reaction with 2.0 M sulfuric acid. (A/A₁, D/D₁, G/G₁) TMB in citrate buffer with α-Fe₂O₃ nanoparticles and no H₂O₂ (control-1), (B/B₁, E/E₁, H/H₁) TMB in citrate buffer with both α-Fe₂O₃ and H₂O₂, and (C/C₁ F/F₁, I/I₁) TMB in citrate buffer with H₂O₂ and no α-Fe₂O₃ (Control-2).

The respective TEM images of the three different shaped nanoparticles and corresponding Lineweaver–Burk plots are provided in Fig. 2. Additional SEM images of hexagonal prism and cube-like α-Fe₂O₃ are presented in Fig. S8, ESI.† We witnessed a rise in V_max values from hexagonal prism (5.0 ± 0.44 nM s⁻¹) (Fig. 2A and B) through cube-like (6.3 ± 0.35 nM s⁻¹) (Fig. 2C and D) to rod (R-1) (Fig. 2E and F), showing the highest maximal reaction velocity (16.5 ± 2.1 nM s⁻¹). On the other hand, the lowest K_m value was shown by the rods (0.257 ± 0.089 mM) followed by the hexagonal prisms (0.768 ± 0.091 mM) and then the cube-like nanoparticles (0.957 ± 0.085 mM). In context to the natural enzymes, V_max and K_m values are the indicators of maximal reaction velocity (i.e. the rate of reaction when an enzyme is saturated with the substrate) and affinity (ability to bind with the substrate. The lower the K_m values, the greater the affinity).

Fig. 2 TEM images and Lineweaver–Burk plots of α-Fe₂O₃ hexagonal prism (A and B), cube-like (C and D), and rod shaped (E and F) nanoparticles used for peroxidase enzyme mimic studies.

When these two traits of the enzyme activity were extrapolated for these three types of α-Fe₂O₃ nanoparticles, it was seen that the V_max increased with increase in the surface area. As the surface area increases from hexagonal prism to rod via cube-like particles, V_max values also increase, showing the highest values for rod and the lowest values for hexagonal prism, suggesting a sound correlation between maximal reaction velocity and surface area. As expected, K_m values showed an inverse relationship with surface area. However, hexagonal prisms showed slightly enhanced affinity with lower K_m value than that of cube-like nanoparticles for unknown reasons, but in terms of activity, cube-like nanoparticles were slightly better. From the results discussed above, it can be concluded that the peroxidase mimic activity of all the three α-Fe₂O₃ nanoparticles was mainly influenced by their surface area. Fig. 3 represents a graphical plot of K_m and V_max values plotted as a function of surface area. The respective V_max, K_m and K_cat values for all the nanoparticles studied are summarized in Table 1, providing a collective account of correlation between shape, size, surface area and the kinetic parameters representing enzyme mimic activity.

Fig. 3 Correlation plot showing V_max and K_m values as a function of surface area for hexagonal prism, cube-like and rod shaped nanoparticles.

Till this point of investigation we observed a sound correlation between enzyme mimic activity and the surface area of nanoparticles. However, the difference in the surface area of rods when compared with the hexagonal prisms and cube-like nanoparticles is not significant enough to explain its exceptionally high activity, i.e. ca. 2–3 times higher activity than those of the cube-like and hexagonal prisms. This clearly indicates that not only the surface area, but other factors also should be responsible for the higher catalytic activity of the rods. This was the most intriguing part of the study, which forced us to further examine the peroxidase mimic activity of rod shaped nanoparticles in detail. To do so, in addition to the already analyzed rod R-1 we chose two more rods (R-2 and R-3) having different surface areas and slight variations in their length. The size of these three rods varied between 250–400 nm in length, and the diameters ranged between 30–35 nm. Surface area increased in the order of R-1 (55 ± 5 m² g⁻¹) < R-2 (82 ± 8 m² g⁻¹) < R-3 (111 ± 10 m² g⁻¹) with R-3 having the highest surface area, while R-1 having the lowest one. In order to verify the ability of R-2 and R-3 to catalyze the peroxidase substrate TMB, they were subjected to similar experimentation as that of R-1. The data obtained were processed as described in Section 3.2. Coloured images of peroxidase mimic activity for all the three rods are provided in Fig. S9, ESI.†TEM images for all the three rods with respective Lineweaver–Burk plots are provided in Fig. 4. The V_max values of R-1, R-2 and R-3 are in the following order: R-1 > R2 > R-3, while the K_m values display a reverse trend: R-1 < R-2 < R-3 with R-1 showing the maximum enzyme mimic activity followed by R-2 and R-3. It is very interesting to note that the V_max and K_cat values of the rods increase with decrease in K_m values and the surface area. Correlation of V_max and K_m values as a function of surface area is plotted in Fig. 5, indicating a deviation in the sequel of the study from earlier outcomes, where the peroxidase mimetic activity was mainly governed by the surface area of nanoparticles. Results shown by our hexagonal prism and cube-like α-Fe₂O₃ nanoparticles were similar to those of the Fe₃O₄ nanoparticles published earlier.⁴ In contrast, the rods having lower surface area (R-1 and R-2) performed as a better peroxidase mimic than the one with higher surface area (R-3), which was truly surprising and “off the trend”.

Fig. 4 TEM images and Lineweaver–Burk plots of α-Fe₂O₃ rod shaped R-3 (A and B), R-2 (C and D), and R-1(E and F) nanoparticles used for peroxidase enzyme mimic studies.

Fig. 5 Correlation plot showing V_max and K_m values as a function of surface area for R-1, R-2 and R-3 nanorods.

Although a conclusive reason is yet to be established for such ‘off the trend’ activity shown by the α-Fe₂O₃ nanorods, other physical features like porosity and pore shape which had not been considered yet, could explain this exceptional trend in catalytic performance to some extent. Generally particle morphology, surface roughness, and surface porosity are the main features influencing the surface area. In the case of α-Fe₂O₃ nanorods, the surface area is mostly credited to the pore shape and number of pores on the particles. An earlier work²² reports that the presence of slit shaped pores along the C-axis results in a larger surface area, while isolated circular pores result in a smaller surface area. If we look at the enlarged TEM images of the rods presented in Fig. 6, it is clearly visible that R-2 and R-3 mainly have slit shaped pores and both of them have a higher surface area compared to R-1, which has large circular pores (4–20 nm) and a lower specific surface area. The reason for R-1 with lower surface area being more active may be related to the circular pore shape, which would provide a favourable space for higher ferrous ions–substrate interaction, resulting in much higher catalytic activity compared to the other two rods with slit shaped pores. In the cases of R-2 and R-3, the difference in length between R-2 and R-3 is not significant but there is a considerable difference in the surface area and catalytic activity. The most likely reason for such behaviour can be explained on the basis of enlarged images of R-2 and R-3 in Fig. 6. Both of the rods mainly possess slit shaped pores, and there are reasonable chances that the number of pores present on R-3 may be higher compared to that of R-2, but the pore size (less than 1 nm) may not be wide enough to facilitate good substrate–ferrous ion interactions. Whereas R-2 may have lesser number of pores, the higher catalytic performance suggests that the pore structure may be wide enough to facilitate better substrate–ferrous ion interactions than that of R-3. From the above mentioned comparison of physical parameters with respect to enzyme mimic activity, it can be speculated that pore shape and structure may also be a contributing factor to the peroxidase mimic activity of rod type materials.

Fig. 6 Enlarged TEM images of R-3 (right), R-2 and R-1 (left) showing the slit and circular shaped pores.

Results from our and earlier studies suggest that if similar investigations are protracted in other enzyme mimicking nanomaterials such as FeS,²³cerium oxide,³ a better understanding of the relation between physical parameters and enzyme mimic activities of nanoparticles may be obtained that may act as guidelines for development of effective nanoparticle-based enzyme mimics. Further study in this regard is under consideration.

4. Conclusions

Current work attempts to explore various facets of hematite α-Fe₂O₃ nanomaterials as an enzyme mimic. The α-Fe₂O₃ nanoparticles with different nanostructures such as hexagonal prism, cube-like, and rod have been demonstrated for the first time as enzyme mimics that possess peroxidase-like activity. The V_max, K_m and K_cat values of the α-Fe₂O₃ reveal that the peroxidase-like activity is mostly governed by the surface area, structure and pore shape of the nanomaterials. Surface area must be a key physical aspect responsible for the enzyme mimic activity. Hematiteα-Fe₂O₃ nanoparticle enzyme mimics having one-dimensional nature act as a more efficient enzyme mimic system than zero or multi-dimensional counterparts. On the other hand, amongst α-Fe₂O₃ nanorods, the peroxidase mimic activity increases with decrease in surface area, which still needs further work for a better understanding. Surface features like porosity and pore shape are also involved in contributing towards enzyme mimic activity in the case of one-dimensional rods. Similar studies on other enzyme mimicking nanomaterials will certainly help in establishing a set of principles which may act as guidelines for development of effective nanoparticle-based enzyme mimetics.

Acknowledgements

This work was supported by Korea University (2011). The authors would also like to thank the Korean Basic Science Institute at Jeonju and Chuncheon for SEM and TEM analyses.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: N₂ isotherms, XRD patterns, length distribution, SEM images and peroxidase enzyme-like activity of different α-Fe₂O₃. See DOI: 10.1039/c1cy00124h

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