Counter Anion-Directed Growth of Iron Oxide Nanorods in a Polyol Medium with Efficient Peroxidase-Mimicking Activity for Degradation of Dyes in Contaminated Water.

Development of nanozymes, which are nanomaterials with intrinsic enzymatic properties, has emerged as an appealing alternative to the natural enzymes with tremendous application potential from the chemical industry to biomedicine. The self-assembled growth of micrometer-sized oxide materials with controlled nonspherical shapes can be an important tool for enhancing activity as artificial enzymes, as the formation of these superstructures often results in high surface area with favorable impact on catalytic activity. Herein, the growth of rod-shaped Fe3O4 microstructures via a one-pot microwave-based method and using a water-poly(ethylene glycol) mixture as a solvent is reported, without the involvement of external shape-directing agents. The precursor metal salt played a key role in the size, shape, and phase selective evolution of iron oxide micro/nanomaterials. Whereas self-assembled microrod superstructures were obtained using Fe(NO3)3 as the metal salt precursor, use of FeCl3 or Fe-acetate as precursors afforded hollow Fe2O3 microparticles and Fe3O4 nanoparticles, respectively. A graphitic layer was deposited on the Fe3O4 surface, imparting a negative surface charge as a result of a high-temperature treatment of poly(ethylene glycol). The rod-shaped Fe3O4 microcrystals show efficient peroxidase-mimicking activity toward 3,3,5,5'-tetramethylbenzidine and pyrogallol as peroxidase substrates with a Michaelis-Menten rate constant (K m) value of 0.05 and 0.52 mM, respectively. The proficient enzyme mimicking behavior of these magnetic superstructures was further explored for the degradation of organic dyes that includes rhodamine B, methylene blue, and methyl orange with a rate constant (k) of 0.038, 0.011, and 0.007 min-1 respectively, using H2O2. This fast and simple method could help to develop a new pathway for differently shaped oxide nanoparticles in a sustainable and economical manner that can be harnessed as nanozymes for industrial as well as biological applications.

Introduction

Iron oxides (n class="Chemical">Fe3O4 or Fe2O3) are one of the most abundant and important minerals on earth. Considering the vast technological applications of these materials in their nanometer dimension, development of synthetic methods with control over size and shape is crucial. A size- and shape-dependent magnetic behavior along with biocompatibility of iron oxide nanoparticles has been harnessed for applications in areas such as ferrofluids, heterogeneous catalysis, magnetic recording media, removal of toxic elements (e.g., As, Pb, and Hg) from water, and several biomedical applications (e.g., gene therapy, immunoassay, contrast enhancement in magnetic resonance imaging, tissue repair, cell sorting, targeted drug delivery for diseases such as cancer, hyperthermia, and DNA separation).[1−12] Particular applications, such as biomedical ones, require the size of the particles to be precisely controlled in the nanometer dimension for effective permeability in vivo.[13,14] However, for several other applications, it has been suggested that microstructures perform better than the corresponding nanostructures.[15] Moreover, shape control is considered as a key for the enhancement of several properties, including catalysis, as the exposed crystal facets and hence atomic arrangement on the facets are known to have a profound effect on its activity.[16−20] Therefore, there has been a great emphasis on the shape-selective growth of self-assembled ordered micrometer size iron oxide superstructures from an assembly of nanocrystals that ensures higher surface area, resulting in enhanced catalytic activity.

Several techniques have been adopted for the development of shape-selective iron oxides that include mechanochemical (electrodeposition, pyrolysis, laser ablation, con class="Chemical">mbustion, etc.) and chemical (sol-gel synthesis, temperature-assisted synthesis, hydrothermal, microwave, reverse micelle, etc.) methods.[21−24] The hydrothermal methods are especially attractive for tuning the morphology of iron oxides, and various shapes like nanorods, cubes, rings, nanospindles, and hollow particles have been reported using this method.[25−29] On the other hand, microwave methods provide a rapid and sustainable synthetic protocol for achieving shape-selective morphology of iron oxides. Recently, Polshettiwar et al. have reported the growth of iron oxides with variable shapes using a template-based method in a cyclohexane-water-pentanol reaction medium using a variety of iron precursors having different solubilities.[30] The polyol method of synthesis, which involves ethylene glycol as a solvent as well as stabilizing agent, has evolved as a soft chemical method for the preparation of a large variety of metal and metal oxide nanomaterials with tunable size, shape, and composition.[31−33] However, the role of counterions in the precursor metal salts on the shape-selective growth of nanomaterials has not been understood clearly. It is recognized that during the growth the inorganic anions themselves might be selectively adsorbed on particular facets and thus greatly impact the final morphology.[34−36] Therefore, it might be possible to obtain shape-selective iron oxides by varying the precursor metal salts in a polyol-based synthesis without using any external templates.

Iron oxide nanomaterials can be used as a low-cost and biocompatible alternative to the natural peroxidases, which are prone to loss of activation in a harsh chemical envn class="Chemical">ironment.[37] Studies of the structural effects of these materials on their enzyme-mimetic behavior may be of high significance in designing materials for practical utility. In this direction, we studied the evolution of self-assembled iron oxide nano/microparticles with different morphologies using a polyol-based microwave synthetic method just by varying the metal salts (Scheme ). Further, we evaluated the peroxidase-mimicking activity of the iron oxides and have observed significant enhancement in the nanozyme activity in the case of rod-shaped superstructures originated in the presence of NO3– as counter anions. Taking advantage of the enhanced peroxidase mimetic behavior of these materials, we have utilized these materials for swift degradation of commercial dyes through a heterogeneous Fenton process that involves the generation of hydroxyl radicals (•OH) by the reaction of hydrogen peroxide and nanoscale iron oxide for the effective destruction of dyes. Overall, a rapid polyol-based microwave-assisted synthetic method has been developed for the generation of shape-selective Fe3O4 microstructures, which shows enhanced nanozyme activity and can be harnessed for effective degradation of organic contaminants.

Scheme 1

Schematic Representation of Counter Anion-Dependent Evolution of Variable Shaped Iron Oxides via a Polyol-Based Microwave-Assisted Method

Results and Discussion

Synthesis and Characterization of Fe3O4 Microrods

Self-assembled n class="Chemical">Fe3O4 microrods were synthesized via a simple microwave-assisted pathway using Fe(III) nitrate as the iron precursor and a mixture of PEG-200–water (PEG:water = 9:1 v/v) as the solvent at 150 °C for 60 min. It is well known that shape-selective synthesis of metal or metal oxide nanoparticles usually requires external templates, which dictates the oriented growth of the crystallites; however, in the present case, the shape selectivity leading to the formation of Fe3O4 microrods could be achieved simply by microwave heating of the metal salt in the polyol medium. The dark brown precipitates thus obtained were analyzed by various spectroscopic and microscopic tools. Scanning electron microscopy (FESEM) studies confirmed the formation of the rod-shaped microcrystals having dimensions of 800 nm width and 3–6 μm length (Figures a and S1). However, clusters of small nanocrystals of an average diameter of 10 ± 5 nm were found decorating the microcrystal surface. The results suggest that small Fe3O4 nanoparticles were probably assembled and grown directionally to form the microparticles. Transmission electron microscopy (TEM) studies also suggested the formation of the Fe3O4 microrods having similar dimensions as obtained from the FESEM studies. Ultrasmall nanoparticles randomly adhering to the microcrystals were clearly visible in the TEM images (Figure b). The HRTEM image of the nanoparticles bound on the microparticle surface showed lattice fringes with interplanar spacing of 0.25 nm that can be attributed to the (311) atomic plane of magnetite (inset: Figure b). Furthermore, the elemental mapping of the microrods reveals the existence of Fe, O, and C in the matrix and all of these three components are spread homogeneously in the microrod framework (Figure c). Energy dispersive x-ray spectroscopy (EDX) studies also validated the presence of carbon along with Fe and O in the matrix (Figure S2).

Figure 1

(a) Scanning electron microscopy (FESEM) image, (b) TEM image (scale bar 1 μm) of Fe3O4 microrods; inset: HRTEM image of nanoparticles decorated on the microrod (scale bar 5 nm), and (c) Elemental mapping of Fe3O4 microrods obtained under microwave condition at 150 °C for 60 min in a water–PEG mixture (1:9) using nitrate salt as a precursor.

(a) Scanning electron microscopy (n class="Chemical">FESEM) image, (b) TEM image (scale bar 1 μm) of Fe3O4 microrods; inset: HRTEM image of nanoparticles decorated on the microrod (scale bar 5 nm), and (c) Elemental mapping of Fe3O4 microrods obtained under microwave condition at 150 °C for 60 min in a water–PEG mixture (1:9) using nitrate salt as a precursor.

The phase characteristics of the microparticles were obtained from X-ray diffraction (XRD) patterns shown in Figure a. Diffraction peaks at 2θ of 18.3, 30.1, 35.5, 43.1, 57.0, and 62.6 could be perfectly indexed to spinal n class="Chemical">Fe3O4 and correspond to (111), (220), (311), (400), (511), and (440) lattices, respectively (JCPDS no. 65-3107). Interestingly, a broad band with maxima at around 2θ of 23.9° was also observed, which could be assigned to the (002) plane of thin carbonaceous layers.[38,39] This suggests that a thin graphitic layer might be deposited on the Fe3O4 nanoparticles. Raman spectroscopy is a potent tool to characterize a distinctive arrangement of crystal structures of carbon. Raman spectroscopy studies showed two additional bands at 1350 and 1590 cm–1 along with characteristic peaks of Fe3O4 in the range of 250–800 cm–1 (Figure b). The bands at 1350 and 1590 cm–1 could be endorsed to the D and G modes of carbon in Fe3O4 microrods matrix, respectively.[40,41] Whereas the G mode is originated from the planar vibrations of sp2 hybridized carbon atoms, the D mode is defect-induced vibration originated from the disordered structure of graphite and shows that the carbon shell is partially graphitized and amorphous in nature. Small molecular weight polymers such as PEG-200 are known as the carbon precursor for the synthesis of carbonaceous materials such as carbon dots under microwave irradiation.[42] Hence, the formation of a thin graphitic layer on the Fe3O4 microrods during the microwave heating of Fe metal precursors using a polyol method is not surprising.[43−46] X-ray photoelectron spectroscopy (XPS) analysis was performed to understand the composition and oxidation state of the Fe3O4 microrods. The full spectrum of the survey scan shows that iron (Fe), oxygen (O), and carbon (C) are present in the sample. As shown in Figure c, two major XPS signals located at BEs of 710.3 and 724.0 eV can be attributed to Fe 2p3/2 and Fe 2p1/2 levels, respectively. These peaks are further deconvoluted to 4 peaks at 710.5, 713.2, 724.0, and 726.4 eV (Figure S3a). The peaks at 710.5 and 724.0 eV confirm the Fe2+ chemical state; on the other hand, peaks at 713.2 and 726.4 eV correspond to Fe3+ in the Fe3O4 phase. A small satellite peak at 719.0 eV confirmed the formation of Fe3O4 phase in the microrods.[47−50] The C 1s core-level spectrum of Fe3O4 microrods could be fitted into three components, i.e., 284.4, 286.2, and 288.4 eV, which correspond to C–C/C–H, epoxy carbon in C-O, and carboxyl groups, respectively (Figure S3b).[51] The results further confirm the presence of a thin carbon layer on the Fe3O4 composite. Deconvolution of the O 1s signal suggested three peaks having binding energies of 529.6, 531.5, and 533 eV, coinciding with lattice oxygen, carbonyl oxygen, and alkoxy oxygen in Fe3O4 microrods, respectively. (Figure S3c).[52] The magnetic property of the Fe3O4 microparticles was investigated using a SQUID magnetometer (Figure d). The M–H curve of the as-prepared material showed zero magnetic coercivity at 300 K, depicting a super-paramagnetic behavior. The saturation magnetization value was found to be 48 emu g–1, which is lower than reported values for bare Fe3O4 nanoparticles. The reduced Ms value of the microparticles might be attributed to the interaction of disordered amorphous carbonaceous layers with the Fe3O4 surface.[53]

Figure 2

(a) XRD spectrum, (b) Raman spectrum, (c) wide range XPS spectrum, and (d) room-temperature magnetization curves as a function of the field (M–H curve) of Fe3O4 microrods obtained under microwave condition at 150 °C for 60 min in a water–PEG mixture (1:9) using nitrate salt as a precursor.

(a) XRD spectrum, (b) Raman spectrum, (c) wide range XPS spectrum, and (d) room-temperature magnetization curves as a function of the field (M–H curve) of Fe3O4 microrods obtained under microwave condition at 150 °C for 60 min in a n class="Chemical">water–PEG mixture (1:9) using nitrate salt as a precursor.

The molecular orientation and local bonding environments of the synthesized n class="Chemical">Fe3O4 particles were further confirmed by FTIR spectroscopy (Figure S4). The FTIR spectrum showed major vibrational peaks at 3440, 2928, 2360, 1628, 1053, 782, and 631 cm–1, which can be owing to the vibrational modes of v(O-H stretching), v(-CH2), v(C–C stretching), v(C=C stretching), v(C-O stretching), v(O-H bending), and v(Fe-O), respectively.[54,55] Further, two prominent peaks at 1427 and 1354 cm–1 were observed, which might arise due to the formation of mono or bidentate complex of carboxylic group of the carbon layer with surface Fe atoms.[56] The results suggested that the Fe3O4 surface was surface-passivated by a carboxyl-functionalized carbon layer. This was further evident in the ζ-potential measurement of the synthesized materials, which showed a value of −21.3 mV, as compared to a value of +15.2 mV for a bare Fe3O4 nanoparticle (synthesized through the well-known coprecipitation method using a mixture of Fe2+ and Fe3+ salts and NaOH under a N2 atmosphere in water).[57] Thermogravimetric analysis (TGA) was employed to investigate the thermal stability of the synthesized material. As shown in Figure S5, a weight loss of 3–4% up to 150 °C could be attributed to the removal of the physically absorbed water. There was further weight loss of 16% probably due to the elimination of functional groups containing labile oxygen from the surface.[41,58] The N2 isotherm, as shown in Figure S6, possesses the type IV isotherm with a small hysteresis loop in the 0.4–1.0 P/P0 range, signifying the existence of mesopores. The Brunauer–Emmett–Teller (BET) surface area was calculated to be 62.0 m2 g–1, and the pore-size distribution from desorption analysis by the Barrett–Joyner–Halenda method was found to be 3.7 nm. Comparing to the small surface area of Fe3O4 nanoparticles,[59] the composite has significantly enhanced surface area, which might be due to the presence of a mesoporous carbon layer on the surface.

Growth Mechanism

The polyol method, which involves the use of n class="Chemical">poly(ethylene glycol) as both a reducing and stabilizing agent, is a well-known strategy for the growth of metal and metal oxide nanoparticles, where the nucleation and growth of the nanocrystallites can be achieved with desired thermodynamic and kinetic control leading to self-assembled nanostructures. Shape selectivity of the nanocrystals is usually achieved by additional shape-directing agents, which are adsorbed preferentially on specific crystallographic planes, resulting in the change of direction and rate of crystal growth. Although the role of various additives on the stability of crystallographic planes is elaborately studied, there are only a handful of reports elucidating the influence of inorganic counterions in shape-selective growth of metal oxide nanoparticles without the involvement of external agents.[60,61]

For the synthesis of the shape-selective Fe3O4 microrods, efficient and controlled heating provided by microwave irradiation was used in a tightly sealed closed vessel and the growth of the microrods was observed using n class="Chemical">Fe(NO3)3 as the starting precursor. Due to the fast kinetics of the nanocrystallite synthesis, it was not possible to elucidate a growth mechanism from time-dependent studies, as the microrod formation was observed even after 15 min of microwave heating (Figure a). However, the decoration of small Fe3O4 nanoparticles on the microrods clearly suggests that small nanocrystallites were initially formed, which gradually self-assembled into a microrod over time. Further, controlled experiments revealed that the amount of water in the solvent mixture played a significant role in the self-assembly process. No Fe3O4 nanoparticles were obtained without water injection. Whereas microrod formation occurred predominantly at a water-to-PEG ratio of 1:9 in the solvent mixture, only a few microrod formations were observed at a water-to-PEG ratio 1:1. On the other hand, only nanospheres were formed when the water-to-PEG ratio was maintained at 9:1 (Figure S7). The results clearly infer the role of PEG in the self-assembled growth of Fe3O4 microrods. Sealed vessel microwave processing allows rapid heating of the reaction mixture, allowing quick occurrence of the nucleation event relieving the solution supersaturation. Further growth of the initially formed nucleates takes place in a high and constant flux of monomers in a diffusion-limited process. The nanoparticles are evolved progressively to higher dimensions in a process where the dissolution and migration of adatoms to selective crystal planes takes place continuously, to minimize the total surface energy (Ostwald ripening). Along with their use as a high boiling point solvent, PEG also can chelate with the surface of metal oxide nanoparticles. When Fe(NO3)3 is dissolved in a 9:1 PEG–H2O mixture, there are two distinct phases: (i) formation of a metal–aqua complex through the coordination of Fe3+ ions with water molecules and (ii) intermolecular hydrogen bridging of H2O with PEG as the majority phase. At an elevated temperature (higher than 100 °C), water is absent at the initial stage and thus the rate of migration of adatoms is highly suppressed. This plays an important role in controlling kinetics of the growth and stimulates anisotropy. The growth rate might be further influenced by a significantly slower diffusion rate of precursor metal salts in PEG, considering the higher viscosity of polyol medium as compared with water.

Figure 3

(a) SEM micrograph of Fe3O4 microrods obtained under microwave condition at 150 °C for 15 min (water–PEG ratio 1:9) and (b) schematic presentation depicting the formation mechanism of Fe3O4 microrods obtained under microwave condition at 150 °C using Fe(NO3)3 precursor salt and water–PEG as a solvent.

(a) SEM micrograph of Fe3O4 microrods obtained under microwave condition at 150 °C for 15 min (n class="Chemical">water–PEG ratio 1:9) and (b) schematic presentation depicting the formation mechanism of Fe3O4 microrods obtained under microwave condition at 150 °C using Fe(NO3)3 precursor salt and water–PEG as a solvent.

In a microwave-based synthesis, the heating rate is quite fast and the reaction temperature is achieved in a minute or two; hence, it is very difficult to perform a kinetic study to understand the growth mechanism and the intermediates involved. When we performed the microwave synthesis at 80 °C keeping all other conditions similar, we obtained a cluster of nanoparticles after 5 min of microwave heating. SEM studies showed the formation of nanoparticles having dimension of 40 ± 10 nm in diameter (Figure S8). X-ray powder diffraction study revealed that the obtained nanoparticles had tetragonal FeOOH (β-n class="Chemical">FeOOH) structure (Figure S9). From these studies, we believe that the formation of the Fe3O4 microrods took place via β-FeOOH nanoparticle intermediates. Thus a plausible formation mechanism can be suggested asIn an aqua–PEG mixture, the initially formed iron aqua complex undergoes dehydration to generate FeOOH nuclei under a microwave treatment. A combination of coarsening and oriented self-aggregation of the initially formed embryos leads to the growth of larger crystals with shape selectivity largely controlled by the Ostwald ripening process. A phase transformation occurred after an extended reaction period. The presence of PEG as a mild reducing agent ensures the reduction of Fe3+ to Fe2+, thus resulting in the formation of Fe3O4 microrods. It can be clearly observed that the Fe3O4 microrods consist of smaller Fe3O4 particles as building blocks and have irregular surface, demonstrating that the Fe3O4 nanoparticles self-assemble into the microrods (Figure b).

It was interesting to note that the self-assembled growth of n class="Chemical">Fe3O4 nanoparticles into one-dimensional microrods occurred without the addition of any shape-selective agents. We assumed that the NO3– counterions might play a critical role in the shape evolution. Therefore, we varied the metal salt precursors in the microwave-assisted polyol process, while keeping all reaction parameters, such as concentration of the metal salt, water-to-PEG ratio, reaction temperature, etc. constant. When FeCl3 was used as the precursor metal salt, hollow Fe2O3 microparticles with an average diameter of 1.1 ± 0.1 μm were obtained. SEM studies (Figure a) reveal the surface configuration of the microspheres, which is not smooth, implying that these microspheres are comprised of small nanoparticles as primary building units and self-assembled to form the larger near spherical aggregates. The formation of internal voids in the microspheres can be clearly seen in cracked microspheres. All diffraction peaks in the hollow microsphere powder XRD spectra (Figure b) could be indexed to the α-Fe2O3 hexagonal phase (JCPDS card no. 33-664). Considering the oxidative etching properties of the halide ions,[62,63] the formation of the complex hollow assemblies with a different phase could be easily understood. When iron acetate was used as the precursor metal salt, only small spherical Fe3O4 nanoparticles with an average dimension of 40 ± 10 nm were obtained. Acetates are known to have high binding affinity for oxide surfaces and hence act as an efficient stabilizer restricting the growth to smaller dimensions. (Figure c). The results suggest that the inorganic anions from the precursor salts were influential in controlling the size, shape, and structural evolution of the micro/nanoparticles.

Figure 4

(a) SEM image of Fe2O3 microparticles obtained using FeCl3 salt as a precursor under microwave irradiation at 150 °C for 60 min in a water–PEG mixture (1:9), (b) XRD spectrum of Fe2O3 microparticles; (c) SEM image of Fe3O4 obtained using Fe(OAc)3 as a salt precursor, and (d) SEM image of Fe3O4 microrods obtained using Fe(NO3)3·9H2O under a hydrothermal treatment at 180 °C for 12 h.

It is well-known that during the crystallization process, the inorganic anions might selectively adhere on particular facets. The FeOOH nucleates initially obtained are stabilized by n class="Chemical">PEG molecules through chelation, thus suppressing the nucleation and growth of nanocrystals in the solution. The aggregation of formed nuclei takes place to eliminate the interfaces and minimize the total energy of the system. NO3– ions are known to have different adsorption strengths on different crystal facets,[64] which might cause faster growth of nanocrystals in the direction with weaker adsorption. Although the exact mechanism for the microrod morphology could not be traced, we believe that a combination of both anion facet coating and stabilization of adatoms by PEG was crucial for growth of various superstructures. This was further confirmed by the fact that when Fe(NO3)3 was hydrothermally treated in a Teflon-sealed autoclave using a PEG–water mixture as a solvent at 180 °C for 12 h, the resultant precipitate consisted of Fe3O4 microrods with an average diameter of 0.5–0.7 μm and several micrometers of length with a much smoother surface as compared with the microwave-based method (Figure d). The formation of the graphitic layer on the nanoparticle surface could be realized as the microwave/hydrothermal treatment of PEG leads to the formation of carbonaceous materials.

Oxidation of Peroxidase Substrates Catalyzed by Rod-Shaped Fe3O4 Microcrystals

In recent years, the enzyme-mimetic activities of metal oxide nanoparticles have been pursued extensively. The natural enzymes are prone to deactivation if the reaction conditions such as pH of the medium or temperature are modulated to some extent. Due to their ease of synthesis, coupled with their stability under harsh reaction conditions, n class="Chemical">metal oxides are considered as an ideal alternative to natural enzymes for various applications.[65−70] The peroxidase-mimicking activity of Fe3O4 nanoparticles is well studied, and there are reports on the enhancement of peroxidase activity depending on the shape of the nanocrystals.[59] The catalytic oxidation of the 3.3,5,5′-tetramethylbenzidine (TMB) was carried out in the presence of H2O2, to assess the peroxidase-like activity of the Fe3O4 microrods, and the progress of the reaction was monitored via UV–visible spectroscopy. As shown in Figure a, the colorless substrate TMB is oxidized in the presence of H2O2 and Fe3O4 microrods to a blue solution, with enhancement in absorption intensity at 370 and 652 nm. In the absence of H2O2, addition of only Fe3O4 microrod to TMB resulted in negligible color variation under similar experimental conditions. These results indicate that the Fe3O4 microrods demonstrate an efficient peroxidase-like behavior toward typical peroxidase substrates like TMB and a blue charge-transfer complex (chromogen) is formed quickly, catalyzed by the Fe3O4 microrods in presence of H2O2. To achieve an optimal response, the effects of pH on the catalytic activities of the Fe3O4 microcrystals were investigated in a series of buffer at varying pH from 2 to 10. The response curves (Figure b) show that the maximum catalytic activity was obtained at pH 4.0, which is very similar to the operating conditions for horseradish peroxidase (HRP).[37,71] To eliminate the role of any leached iron in the catalytic reaction, the iron contents of the supernatants after removal of the microrods by centrifugation were measured by inductively coupled plasma atomic emission spectroscopy and the amount of Fe ions was negligible in the pH range of 3–6. The catalytic activity of any leached iron ions was evaluated for the catalytic reaction at pH 4.0. For this, the Fe3O4 microrods were incubated in an acetate buffer at pH 4.0 for 30 min and the supernatant solution after removal of the microrods was used to monitor the oxidation of TMB. The original colorless solution remained colorless, confirming that no leached Fe ions were involved in the catalytic process, as the concentration of leached ions was much lower than the amount required for typical Fenton reactions. Thus, a 100 mM acetate buffer solution at pH 4.0 was selected as the optimal reaction medium for all subsequent studies.

Figure 5

(a) UV–visible spectra of TMB-Fe3O4, TMB-Fe3O4-H2O2, and TMB-H2O2 reaction system; (b) pH-dependent relative activity of Fe3O4 at room temperature; (c) concentration-dependent relative activity of Fe3O4 at 25 °C using H2O2 as a substrate and (d) TMB as a substrate (the maximum point in the curve was set as 100%); (e) UV–visible steady-state kinetic study of Fe3O4 at fixed H2O2 concentration (4 mM) while TMB concentration was varied; (f) UV–visible steady-state kinetic study of Fe3O4 at fixed TMB concentration (0.1 mM) and variable H2O2 concentration; (insets of (e) and (f)) double-reciprocal plots of Fe3O4 activity at a fixed concentration of one substrate (TMB and H2O2) versus varying concentration of the other.

The dependence of the peroxidase-mimicking activity of Fe3O4 microrods on various parameters, such as concentration of n class="Chemical">TMB and H2O2 and amount of the catalyst, was also carefully monitored by observing the absorption peak at 652 nm (Figure c,d). The maximum catalytic efficiency was achieved at 0.1 mM of TMB, whereas in the case of H2O2, a higher concentration of H2O2 was required to attain the maximum peroxidase-like activity. This indicates that the catalytic activity of the microcrystals is more prominent at high concentrations of H2O2 as compared with HRP. At higher concentration of TMB in the reaction medium, the peroxidase activity was weakened, probably due to sufficient catalytic surface capping at a particular concentration, thus inhibiting the attachment of excess substrates onto the surface. The maximum catalytic activity of the Fe3O4 microrods was achieved at a H2O2 concentration of 1000 mmol/mL, which is 105 times that of TMB. A catalyst concentration as low as 8 μg mL–1 was used to monitor the kinetic parameters.

The steady-state kinetic assays were performed in the TMB-H2O2-Fe3O4 reaction system at room temperature, and the catalytic parameters were evaluated by correlating the absorbance data with the Michaelis–Menten equation (eqn).where Km is the Michaelis constant and ν, Vmax, and [S] correspond to the rate of conversion, maximal velocity (or the maximal conversion rate), and substrate concentration, respectively.

A typical Michaelis–Menten kinetics model was validated during the TMB-H2O2 reaction catalyzed by the n class="Chemical">Fe3O4 microrods, as observed from the initial absorbance against time plots (Figure e,f). The slopes of these plots are evaluated as initial reaction rates at variable substrate concentrations. The catalytic parameters of Km and Vmax were obtained by plotting the reaction rate against concentration and following nonlinear regression using the Michaelis–Menten equation. The Km value of Fe3O4 microrods with TMB as a substrate was calculated to be 0.05 mM, which is significantly lower compared with the reported value of 3.7 mM in the case of native horseradish peroxidase. This indicates a stronger affinity of Fe3O4 microrods for TMB compared with HRP (Table S1).[27,37,72] The higher peroxidase-like activity of the Fe3O4 microrods was consistent for H2O2 as a substrate also (0.09 mM), with a lower Km value. The high peroxidase activity of the microrods can be correlated to the presence of large number of Fe2+ and Fe3+ ions on their surface, compared with only one iron in HRP. For comparison, we also evaluated the peroxidase activity of Fe3O4 nanoparticles synthesized using Fe(OAc)3 as a salt precursor and Fe2O3 hollow microparticles obtained using FeCl3 as the precursor metal salt, using TMB as the substrate. From the plot of the reaction rate vs concentration of TMB and using the Michaelis–Menten kinetic model, Km values were calculated to be 0.15 and 0.31 mM for Fe3O4 nanoparticles and Fe2O3 hollow microparticles, respectively (Figure S10). From the results, it is evident that the Fe3O4 microrods showed superior peroxidase-mimicking activity for TMB, compared with the Fe3O4 nanoparticles or hollow Fe2O3 microparticles. The graphitic layer on the Fe3O4 particles also might contribute to the enhanced peroxidase-mimicking activity of the microrods, as several carbonaceous nanomaterials are known to demonstrate peroxidase-mimicking properties.[68]

It is well-established that peroxidase mimics catalyze the decomposition of H2O2, leading to efficient generation of n class="Chemical">hydroxyl radicals (•OH); therefore, a fluorescent probe was used to track the formation of •OH radicals catalyzed by Fe3O4 microrods. The experiment was performed using weak fluorescent terephalic acid as probe, which generates a highly fluorescent hydroxyl terephthalic acid as a product when reacted with •OH radicals (Figure a).[73,74] In the presence of Fe3O4 microrods and H2O2, the free radical generation was clearly evident, as exhibited by a dramatic increase in fluorescence (Figure b). As the reaction progressed, the fluorescence intensity of terephalic acid enhanced at 440 nm. These results indicated that the peroxidase-mimicking activity of Fe3O4 microrods was mainly due to their potential to generate •OH radicals during the course of reaction.

Figure 6

(a) Schematic presentation of terephthalic acid catalytic oxidation by hydroxyl radical and (b) bar plot of the fluorescence spectrum at 440 nm at different time intervals as a result of terephthalic acid oxidation by Fe3O4 and H2O2. Inset: Normalized fluorescence spectra of the oxidation of terephthalic acid solution containing terephthalic acid, Fe3O4, and H2O2.

(a) Schematic presentation of terephthalic acid catalytic oxidation by n class="Chemical">hydroxyl radical and (b) bar plot of the fluorescence spectrum at 440 nm at different time intervals as a result of terephthalic acid oxidation by Fe3O4 and H2O2. Inset: Normalized fluorescence spectra of the oxidation of terephthalic acid solution containing terephthalic acid, Fe3O4, and H2O2.

Kinetic Study of Pyrogallol

The peroxidase-mimicking activity of the as-obtained Fe3O4 microrods was further evaluated for the oxidation of n class="Chemical">pyrogallol in the presence of H2O2, which forms a yellow purpurogallin complex. Upon addition of Fe3O4 microcrystals (20 μg mL–1) and H2O2 to a solution of pyrogallol in phosphate buffer (pH 7.4), the colorless solution turned yellow, with the appearance of a new absorption peak at 420 nm, signifying catalytic oxidation of the peroxidase substrate (Figure S11a). The reaction was monitored at different time intervals by varying the concentration of pyrogallol and H2O2. The catalytic reaction was completed after 30 min, and the absorbance at 420 nm remained stable. The time-dependent formation of the purpurogallin complex was also monitored for the Fe3O4 nanoparticles and Fe2O3 hollow microparticles, which showed relatively lower catalytic activity compared with the Fe3O4 microrods (Figure S11b). Similar to TMB, the oxidation of pyrogallol also follows Michaelis–Menten kinetics. Studying the kinetics at variable pyrogallol concentrations and keeping the H2O2 concentration constant, a Lineweaver–Burk plot was obtained for catalysis using Fe3O4 microrods, which showed a linear relationship (Figure a,b). From the Lineweaver–Burk plot, the Michaelis–Menten constant Km was evaluated. The calculated Km value of 0.52 mM for pyragallol is lower than that of the natural enzyme HRP (0.81 mM), indicating that the Fe3O4 catalysts have higher affinity toward the substrate.[75] The Km value was obtained from the Lineweaver–Burk plot at variable H2O2 concentrations, while keeping the pyrogallol concentration fixed at 17 mM.

Figure 7

(a) UV–visible steady-state kinetic study of Fe3O4 at a fixed H2O2 concentration (40 mM) while pyrogallol concentration is varied and (b) UV–visible steady-state kinetic study of Fe3O4 at fixed pyrogallol concentration (10 mM) and variable H2O2 concentration; (insets c and d) double-reciprocal plots of Fe3O4 activity at a set concentration of one substrate (pyrogallol and H2O2) compared with different concentrations of another.

Degradation of Dye Pollutants Using Fe3O4 as Catalyst

Organic dyes are predominantly used in several industries, such as photographic printing, textile, tannery, paper-pulp, and paints, and are mixed with soil and water, causing severe damage to the envn class="Chemical">ironment. To get rid of these hazardous pollutants, various processes, such as ozonation, chlorination, adsorption, ultrafiltration, electrochemical processes, photodegradation using photocatalysts, and advanced oxidation processes such as Fenton reactions are used as methods for waste-water treatment.[76] H2O2 is considered to be a green oxidant that behaves as resource of reactive oxygen species. Chemical oxidation routes generate strong oxidizing species such as hydroxyl radical (•OH), which can be used for efficient removal of organic dye contaminants. However, due to low decomposition of H2O2 at room temperature, catalysts are often required for the sustained formation of •OH radicals. A combination of nanocatalysts and H2O2 in a single process could function as an attractive substitute for dye removal.

Encouraged by the high peroxidase-mimicking activity of Fe3O4 microcrystals, we further examined the activity of n class="Chemical">Fe3O4 microrods for the degradation of common organic dyes. Considering the fact that the overall surface charge of the microcrystals was negative, we first studied the degradation of cationic rhodamine B as a model contaminant. The degradation of the dye was visually observed in a mixture of Fe3O4 catalyst, H2O2, and rhodamine B in aqueous medium, and complete decolorization occurred within 120 min at room temperature. The absorption of the dye gradually decreased with increasing Fe3O4 concentration, indicating that the dye degradation efficiency was highly dependent on the nanocatalyst concentration (Figure S12). To optimize the reaction conditions, a concentration-dependent study was performed by varying the concentration of H2O2 from 0–50 mM (Figure S13). At a lower concentration of H2O2, a slower degradation of rhodamine B was observed. As the concentration was increased to 50 mM, the rate of degradation increased, suggesting that a H2O2 concentration of 50 mM was optimal for dye degradation. Further, degradation of rhodamine B was studied at different pH. Figure S14 clearly shows that the maximum rhodamine degradation efficiency was found at pH 4, a similar pH range at which the maximum peroxidase activity was obtained. The quick degradation and a gradual reduction in the prominent absorption peak intensity at 553 nm were further observed during the time-dependent UV–visible spectroscopy studies. A linear correlation between the reaction time and ln(Ct/C0) could be ascertained (Figure a), suggesting that a pseudo-first-order pathway was followed during the dye degradation reaction with a rate constant of 0.038 min–1. Control reactions were performed in the absence of the catalyst or H2O2, and no initiation of the dye degradation occurred even after prolonged time, suggesting that a combination of Fe3O4 microcrystals and H2O2 was essential for the degradation reaction. The excellent performance of Fe3O4 microcrystals for the rhodamine B degradation could be ascribed to a combination of the high surface area of the microcrystals and efficient peroxidase activity.

Figure 8

Catalytic degradation of dyes with H2O2 on Fe3O4 microrods as catalysts: (a) time-dependent absorbance changes at 553 nm for rhodamine B, (b) time-dependent UV–visible spectra of an aqueous solution of rhodamine B; (c, d) time-dependent absorbance changes at 663 nm for MB and 463 nm for MO, (e) dye removal efficiency in the absence or presence of H2O2; and (f) repeating utilization rate of catalyst after the degradation procedure is repeated five times.

From the time-dependent kinetics studies, it was apparent that no shift in the absorption maxima at 553 nm was observed in the initial 60 min (Figure b) and only a continuous reduction in the absorbance was observed. It has been reported that N-de-ethylation and cleavage of the chromophore are two competitive ways of degradation of n class="Chemical">rhodamine B, followed by other reactions, such as hydroxylation, aromatic ring opening, and mineralization. In the case of N-de-ethylation, auxochromic properties of N-ethyl moieties lead to a hypsochromic shift in the absorption maxima.[77] On the other hand, only a reduction in the absorption peak at 553 nm is observed during chromophore cleavage. In the initial stage of the RhB degradation under the present reaction conditions, only a reduction in λmax at 553 nm occurred, suggesting that the chromophore cleavage was the favored pathway for the degradation. The concentration of H2O2 was much higher than rhodamine B in the reaction medium, and most of the N-de-ethylation intermediates formed during the reaction might be cleaved immediately by the generated hydroxyl radicals. Toward the later stages of RhB degradation, the absorption maxima shifted to lower wavelengths by 5 and 11 nm, after 60 and 90 min, respectively. However, any significant effect of the absorption shifting on the reaction kinetics can be neglected, as less than 10% of the original RhB concentration was left at that stage. On the basis of these studies, a synergistic mechanism for the degradation of rhodamine B in H2O2–Fe3O4 system is proposed. H2O2 molecules are adsorbed on the surface of Fe3O4 followed by activation of H2O2 to generate reactive oxygen species (•OH radicals) by the surface Fe(II)/Fe(III) atoms. The •OH radicals then cleave the dyes adsorbed on the surface or diffuse into the solution to degrade the dye molecules near the Fe3O4/solution interface.

The degradation of other commonly used dyes, such as methylene blue (MB) and n class="Chemical">methyl orange (MO), was further monitored using the Fe3O4-H2O2 catalytic system to ascertain the scope of the present method in waste-water treatment (Figure c–e and S15). UV–visible spectroscopy studies were performed to monitor the decay in the concentrations of MB and MO in the solution by observing absorbance at λmax 663 and 463 nm, respectively. In comparison with the degradation of rhodamine B (degradation efficiency DE = 98% and k = 0.038 min–1), the degradation was moderate for MB (DE = 77% and k = 0.011 min–1) and MO (DE = 60% and k = 0.007 min–1), under similar reaction conditions (0.5 mg mL–1 of Fe3O4 microrods, 50 mmol L–1 of H2O2 and 0.1 mmol of dye after 2 h at room temperature). It is evident that RhB was degraded faster than MB, although they are both cationic in nature. The carboxyl group in rhodamine B might influence the formation hydroxyl radicals and facilitate their attack on the dye molecules through hydrogen bonding.[77,78] MO is an anionic dye and possibly is lowly adsorbed on Fe3O4 surface due to the electrostatic repulsion, severely impacting its degradation.

Catalyst reusability and stability are regarded as important parameters for practical catalytic applications. We compared the dye removal activity of the fresh and recovered Fe3O4 catalysts toward n class="Chemical">RhB, and a catalyst concentration of 0.5 mg mL–1 was used. After every cycle, the catalyst is directly withdrawn from the solution by centrifugation followed by thorough washing and redispersed in distilled H2O for the next cycle of catalysis by adding RhB and H2O2. This catalyst retained good activity (82%) even after reuse five times, suggesting high stability of the catalyst even in the presence of a large amount of H2O2 (Figure f).

To verify whether Fe3O4 microrods can be employed to treat envn class="Chemical">ironmental water, tap water and lake water were collected from an industrial area (Pithampur, Indore) and used as the practical sample (Figure S16). This was kept for some time and then filtered before being spiked with rhodamine B as the model pollutant. When Fe3O4 microrods and H2O2 were added to this dye containing water, effective discoloration of the solution occurred within a short time (90 min) due to the degradation of rhodamine B. Even in the lake water containing a mixture of dyes (rhodamine B, MO, and MB), the Fe3O4 microrods could effectively degrade all dyes in the presence of H2O2. Therefore, the Fe3O4 microrods can be employed as a promising nanocatalyst for the environmental waste-water treatment.

Conclusions

A novel microwave-based synthetic methodology has been developed for the rapid self-assembled growth of n class="Chemical">Fe3O4 microrods using polyol method without the aid of any external shape-directing molecular templates. The growth of the size, shape, and morphology-selective micro/nanostructures was highly dependent on the iron precursor salt used for the synthesis, demonstrating the role of counterions in directing the growth mechanism. The microwave treatment of the iron precursors in a polyol medium also led to the growth of a thin carbonaceous layer on the Fe3O4 microcrystals. The Fe3O4 microrods showed excellent peroxidase-mimicking activity against substrates such as TMB and pyrogallol, demonstrating their high capability for applications in environmental remediation in presence of H2O2. This property was taken advantage of for the degradation of common organic pollutants, such as cationic and anionic dyes, with high efficiency. The method presents a simple, one-step synthesis of magnetic microrods with high stability (negligible iron leaching, no phase transformation after reaction), large surface area, and the possibility of magnetic separation, which can be harnessed for several other technological applications, such as degradation of other organic contaminants, photocatalysis, etc.

Experimental Section

Materials

Fe(NO3)3·9H2O, FeCl3·3H2O, and Fe(C2H3O2)2 were purchased from TCI chemicals. Poly(ethylene glycol) 200 (PEG-200), methyl orange (MO), rhodamine B (RhB), methylene blue (MB), and terephthalic acid were obtained from Sigma-Aldrich. 3,3,5,5′-Tetramethylbenzidine (TMB), 1,2,3-trihyroxybenzene (Pyrogallol), and hydrogen peroxide (30%) were obtained from Sisco Research Laboratories (SRL), India. Sodium acetate trihydrate, acetic acid, and NaH2PO4 were purchased from Merck Ltd, India. Ultra-pure water obtained from the Milli-Q system was used in all experiments.

Characterization

UV–visible absorption spectra and kinetic studies were recorded at room temperature on a Varian UV–visible spectrophotometer (Carry 100 Bio). The infrared spectrum (IR) was recorded in a Bruker Tensor 27 FTIR spectrometer in the transmission mode using KBr pellets to prepare the samples. Powder X-ray diffraction spectra (XRD) of the as-prepared sample were measured by a Rigaku Smart lab X-ray diffractometer with Cu Kα radiation (λ = 0.154 nm) in the range of 2θ of 10–80°. For scanning transmission electron microscopy (SEM) studies, samples were prepared by dropcasting on a ITO surface and the images were recorded on a Supra 55 Zeiss apparatus field emission scanning electron microscope, with an energy dispersive X-ray (EDX) attachment. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were recorded using a JEOL JEM-2100 microscope (accelerating voltage of 200 kV). To prepare the samples for TEM testing, the sample solutions were cast on the coated copper grid and dried at room temperature. Emission spectra were recorded using a fluoromax-4p fluorometer from Horiba (model FM-100). The CEM discover Microwave reactor was used for the synthesis of materials. A Raman spectrum was recorded using the Jobin Yvon Horiba LABRAM-HR micro Raman system with a 632.8 He-Ne laser beam attachment. Magnetic property was investigated using an Ever Cool 7 Tesla SQUID Magnetometer. The surface area assessment of Brunauer–Emmett–Teller (BET) was performed on Auto-sorb iQ, version 1.11 (Quantachrome Instruments). X-ray photoelectron spectroscopy (XPS) was performed using the Mg Kα radiation photoemission tool PHI 5000 Versa Prob II, FEI Inc.

Synthesis of Fe3O4 Micro/Nanoparticles with Controlled Morphology

Rod-shaped iron oxide microcrystals were obtained by dissolving n class="Chemical">Fe(NO3)3·9H2O (196 mg, 80 mM) in a PEG-200–water mixture (PEG:water ratio 9:1, total volume 5 mL). The mixture was then transferred into a screw-mount glass tube and subjected to 150 W, 200 psi, and 150 °C microwave radiation for 60 min. This resulted in a dark brown dispersion of Fe3O4. The precipitate was cleaned with water and ethanol several times after cooling to eliminate impurities. For the synthesis of hollow microspheres and nanospheres, FeCl3·3H2O (80 mM) and Fe(C2H3O2)2 (80 mM) were used as iron precursors, respectively, keeping all other synthetic parameters similar. For the hydrothermal synthesis of Fe3O4 nanorods, Fe(NO3)3·9H2O (80 mM) precursor salt was dissolved in 20 mL of the PEG-200–water mixture (the PEG:water ratio was 9:1 v/v) and was transferred in a Teflon-sealed autoclave, which was kept at 180 °C for 12 h.

Peroxidase-Mimicking Activity Studies

The peroxidase-like activity of the synthesized Fe3O4 micn class="Chemical">rostructure was studied for the oxidation of TMB as a substrate. Kinetic experiments were conducted in time-dependent mode using a UV–visible spectrophotometer to monitor absorbance at 652 nm. Experiments were carried out using 20 μL of a Fe3O4 microrod stock solution (1 mg mL–1) in a total volume of 2.5 mL of sodium acetate buffer solution (100 mM acetate buffer, pH 4.0). TMB (0.1 mM) was used as a substrate and H2O2 concentration was 4 mM, unless stated otherwise. The steady-state kinetic measurements were performed under the optimal reaction conditions by varying the TMB concentration from 0.01 to 0.2 mM at a fixed concentration of 4 mM H2O2. Similarly, the kinetic analysis of Fe3O4 with H2O2 as the substrate was performed by using a fixed concentration of 0.1 mM TMB and varying the amount of H2O2 (0.05–0.4 mM ). The effect of pH (2–10), TMB concentration (0.01–0.5 mM), and H2O2 concentration (0.01–2000 mM) on the catalytic activity of Fe3O4 microcrystals was also investigated.

The kinetic studies of pyrogallol oxidation were carried out in a pH 7.4 n class="Chemical">phosphate buffer solution at room temperature. The concentration of pyrogallol was varied while maintaining a constant amount of Fe3O4 catalyst (20 μg mL–1) and H2O2 concentration (40 mM). The Michaelis–Menten constant (Km) was calculated using Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation.

Detection of Hydroxyl Radicals

In a typical reaction process, 1.0 mg of Fe3O4 microrods was dispersed via sonication in 5.0 mL of n class="Chemical">sodium acetate buffer (pH 4.0) solution containing 50 mM H2O2, followed by addition of 1 mM terephthalic acid and 3 mM NaOH. After incubation, the suspension was centrifuged at different time intervals; at an excitation wavelength of 315 nm, the supernatant was collected for fluorescence measurement.

Dye Degradation

For the dye degradation studies, 0.5 mg mL–1 Fe3O4 microrods were dispersed into 20 mL of n class="Chemical">rhodamine B stock solution (0.1 mmol L–1) prepared in acetate buffer (pH 4.0). After preadsorbtion at room temperature for 5 min, the absorption of rhodamine B was measured by UV–visible spectroscopy and considered as C0 (initial concentration). Addition of 50 mM H2O2 into the above solution initiated rapid catalytic degradation of rhodamine B. About 2.5 mL of the solution was taken out from the reaction mixture at an interval of 20 min, and the suspended Fe3O4 particles were removed either by centrifugation at 8000 rpm for 2 min or by magnetic separation. The absorption of the supernatants was recorded on a UV–visible spectrophotometer. Similarly, the dye removal efficiency of Fe3O4 microrods was investigated by time-dependent measurement of absorption maxima of MO (0.1 mM) and MB (0.1 mM) dyes in aqueous medium under similar reaction conditions. We also examined the effect of pH, concentration of catalyst, and H2O2 concentration (0–50 mM) on the catalytic efficiency of dye degradation.