1 Introduction

Enzyme mimics are an upcoming area of research due to their high stability, cost effectiveness, tunable catalytic properties and easy handling and storage in comparison with natural enzymes. They have gathered attention due to their immense commercial potential over their natural counterparts. Although natural enzymes possess efficient catalytic activity, high selectivity and substrate specificity, but their efficiency and stability is severely affected by environmental conditions, denaturation and digestion, thus limiting their commercial applications [1, 2]. Moreover, they are difficult to prepare, purify and store, thus making artificial enzymes preferable over the natural ones. Nowadays they have been applied in the area of environmental protection, chemical/biological sensors, cancer treatment, anti-fouling systems, in pharmaceutical and fuel industries etc. [3,4,5]. The basic approaches to design the artificial enzymes are either to reproduce enzyme active sites or mimic the chemical reaction i.e., chemical transformation of reactants to products without copying the structure of enzyme active sites.

A superoxide (\(\hbox {O}_{2}^{-}\)) anion produced from one electron reduction of molecular oxygen plays a major role in oxidative stress. It also affects the production of other reactive species like peroxynitrite (ONOO–) and its degradation products (OH, \(\hbox {CO}_{3}^{-}\) and \(\hbox {NO}_{2}\)), \(\hbox {H}_{2} \hbox {O}_{2}\), lipid alkoxyl (RO) and peroxyl (\(\hbox {RO}_{2}\)) radicals. Excess of these reactive species results in redox imbalance leading to excessive immune responses, inflammation and various diseases such as complications of diabetes, stroke, reperfusion injury and rheumatoid arthritis [6]. \(\hbox {H}_{2} \hbox {O}_{2}\) resulting from one-electron reduction of superoxide is a dominant signalling molecule. Thus, endogenous antioxidants are essential to maintain reactive species at nanomolar levels. The superoxide dismutase (SOD) family comprises MnSOD and FeSOD located in the mitochondrial matrix and chloroplasts respectively and Cu–Zn SOD is located in the mitochondrial intermembrane space, extracellular space and cytosol. This metalloenzyme eliminates the superoxide anion by catalysing its dismutation i.e. disproportionation to molecular oxygen and hydrogen peroxide, and thus acts in primary internal anti-oxidant defence of the body [7]. However, its use as an anti-oxidant is not only limited to biological reactions but also to various fields like food processing, medical applications, cosmetics and plant industries [8,9,10]. This encouraged the development of synthetic SOD mimic as an anti-oxidant. To date, a wide range of synthetic molecules like manganese and porphyrin based complexes have been explored as SOD mimics [11].

In the last few decades, the potential use of nanoparticles (NPs) has been studied as enzyme mimics due to their high surface to volume ratio [12,13,14]. To date a wide range of NPs have been exploited for their enzyme mimic activity [15,16,17]. Among them, magnetic NPs have proved a powerful tool as enzyme mimics for applications in the field of environmental chemistry, medicines and biotechnology [17,18,19,20]. SOD mimic activity of manganese complexes has been widely studied due to their high stability and low toxicity [21]. However, to the best of our knowledge no work has been performed on SOD mimic activity of ferrite NPs. Thus, in the present work, SOD mimic activity of spinel ferrite \(\hbox {MFe}_{2} \hbox {O}_{4}\, \hbox {NPs}\) (\(\hbox {M} = \hbox {Mn}\), Co and Cu) was studied. The effect of changing a metal ion in a spinel lattice on SOD mimic activity was observed and detailed investigation on the effect of reaction conditions on activity was recorded. Kinetic studies were performed and kinetic parameters were determined using the Michaelis Menten equation.

2 Experimental

2.1 Materials

Chemicals used viz., \(\hbox {Mn} (\hbox {NO}_{3})_{2} {\cdot }\hbox {6H}_{2} \hbox {O}\), \(\hbox {Co} (\hbox {NO}_{3})_{2}{\cdot } \hbox {6H}_{2} \hbox {O}\), \(\hbox {Cu} (\hbox {NO}_{3})_{2} {\cdot } \hbox {6H}_{2} \hbox {O}\), \(\hbox {Fe} (\hbox {NO}_{3})_{3} {\cdot } \hbox {9H}_{2} \hbox {O}\), citric acid, \(\hbox {NH}_{4} \hbox {OH}\), pyrogallol and ethylene diaminetetraacetic acid (EDTA) were of AR grade. The pH of reaction solution was adjusted using 0.1 N HCl and 0.1 N NaOH. Double distilled water was used to prepare the solutions.

2.2 Instrumentation

X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FT-IR) analyses were performed with a Panalytical X’pert Pro using \(\hbox {CuK}_{\upalpha }\) radiation (\(\lambda = 1.5404 \, {\AA }\)) and a Thermo Nicolet 6700 FT-IR Spectrometer. Scanning electron microscopy of ferrite nanoparticles (NPs) was performed by employing Hitachi S-3400 N and EDX on a Thermo Noran System SIX respectively. BET isotherms were analysed using a Quantachrome Nova-1000 surface analyser under liquid nitrogen atmosphere. The measurements of enzyme mimic activity were performed using a UV-1800 Shimadzu Double-beam Spectrophotometer.

2.3 Synthesis of ferrite NPs

Ferrite NPs were synthesized using a sol–gel method [22]. Stoichiometric amounts of metal nitrates were dissolved in double distilled water, followed by the addition of an equimolar amount of citric acid as a chelating agent to the reaction mixture. Ammonia solution was added slowly to adjust the pH of the reaction mixture to 8.0 and the mixture was continuously stirred at \(60{-}70{^{\circ }} \hbox {C}\). After 3 h, the sol was transformed into black gel which was dried at \(100{^{\circ }} \hbox {C}\) in an oven for 8 h. After drying, its volume expanded four times. The dried gel was calcined for 3 h at \(300{^{\circ }} \hbox {C}\) to obtain the final thermolysis product.

Fig. 1
figure 1

FT-IR spectra of (a) \({\hbox {MnFe}}_{2} {\hbox {O}}_{4}\), (b) \({\hbox {CoFe}}_{2} {\hbox {O}}_{4}\) and (c) \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs.

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Fig. 2
figure 2

XRD pattern of (a) \({\hbox {MnFe}}_{2} {\hbox {O}}_{4}\), (b) \({\hbox {CoFe}}_{2} {\hbox {O}}_{4}\) and (c) \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs.

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2.4 SOD like activity of ferrite NPs

Ferrite NPs were tested for SOD mimic activity using the Marklund and Marklund method at \(25{^{\circ }} \hbox {C}\) [23]. SOD/SOD mimics catalyse the dismutation of the superoxide anion (formed during pyrogallol auto-oxidation) into hydrogen peroxide. The resulting oxidized product showed an absorbance maximum (\(\lambda _{\mathrm{max}}\)) at 420 nm. Lesser the value of absorbance, more the dismutation of the superoxide anion, and greater is the SOD mimic activity. The effect of the presence of \(\hbox {MFe}_{2} \hbox {O}_{4}\, \hbox {NPs}\) on the absorbance of the reaction mixture was observed. The activity of \(\hbox {MFe}_{2} \hbox {O}_{4}\) \(\hbox {NPs}\) was compared on the basis of the absorbance value when experiments were carried out at different pH values, temperatures, catalysts and substrate concentrations to evaluate the effect of various parameters.

Table 1 XRD parameters of ferrite NPs.
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Fig. 3
figure 3

SEM–EDX micrographs of (a) \({\hbox {MnFe}}_{2} {\hbox {O}}_{4}\), (b) \({\hbox {CoFe}}_{2} {\hbox {O}}_{4}\) and (c) \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs.

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Fig. 4
figure 4

BET isotherms of ferrite NPs (a) \({\hbox {MnFe}}_{2} {\hbox {O}}_{4}\), (b) \({\hbox {CoFe}}_{2} {\hbox {O}}_{4}\) and (c) \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs.

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The SOD mimic activity was assayed in a spectrophotometric cuvette having a reaction volume of 3 ml containing 2 ml (6 mM) pyrogallol and 1 ml (6 mM) EDTA. Both the contents were mixed and pH of the reaction mixture was adjusted to optimize the value of 8.2 using 0.1 N NaOH solution. The reaction started on adding 1 mg ferrite NP into the solution. The mixture was subjected to atmospheric oxygen for 3 min and the colorimetric analysis was performed spectrophotometrically. A reaction mixture without ferrite NPs was used as blank. The activity of enzyme mimic is expressed in activity unit (A.U.) where 1 A.U. is equivalent to 50% inhibition of pyrogallol oxidation as compared to blank. For steady-state kinetic parameters of ferrite NPs, under optimum conditions i.e. \(\hbox {pH} = 1\) with a dose of 2 mg of NPs for 3 min, the absorbance of the reaction mixture was noted with varying concentrations of the substrate. The reactions were monitored using time scan mode at 420 nm. The absorbance data were fitted in the Michaelis–Menten equation as described below

$$\begin{aligned} v=\frac{v_{\mathrm {max}} [S]}{K_{\mathrm{m}} + [S]} \end{aligned}$$
(1)

where v is the rate of the reaction, \(v_{\mathrm{max}}\) is the maximum rate of the reaction and [S] is the substrate concentration. \(K_{\mathrm{m}}\) is the Michaelis constant which indicates the affinity of the enzyme towards the substrate and is equivalent to the substrate concentration at which the rate of conversion is half of \(v_{\mathrm{max}}\). Lower the value of \(K_{\mathrm{m}}\), higher the enzyme mimic activity. The values of \(K_{\mathrm{m}}\) and \(v_{\mathrm{max}}\) were calculated by plotting the Lineweaver–Burk plot between 1 / [S] and 1 / v. The intercept on x- and y-axes gave values of \(K_{\mathrm{m}}\) and \(v_{\mathrm{max}}\), respectively.

3 Results

3.1 Characterization

In FT-IR spectra of ferrite NPs (figure 1) the peaks appeared in the range of \(548.36{-}576.18 \, {\hbox {cm}}^{-1}\) and \(3434.42{-}3774.69 \, {\hbox {cm}}^{-1}\) that corresponded to the vibration of the metal oxygen (M–O) bond in the tetrahedral sites and O–H stretching of adsorbed water molecules respectively [24]. The XRD patterns of ferrite NPs (figure 2) showed well defined peaks in the crystallographic planes of (220), (311), (400), (511) and (440) which were confirmed by matching with ASTM Data Card no. 10-319, 3-0865 and 25-283. XRD parameters and the particle size calculated from Scherrer’s formula are reported in table 1.

SEM–EDX of NPs using a point and shoot method was recorded at 15 kV at different points and EDX spectra with elemental composition are shown in figure 3. The spectra showed the relative effective atomic concentrations of different constituents in different ferrite NPs (peak of C due to carbon stub). The surface morphology was studied using SEM micrographs and was found to be porous.

Nitrogen adsorption and desorption measurements of the samples were used to evaluate porosity and the specific surface area (figure 4). The BET surface area of \(\hbox {MnFe}_{2} \hbox {O}_{4}\), \(\hbox {CoFe}_{2} \hbox {O}_{4}\) and \(\hbox {CuFe}_{2} \hbox {O}_{4} \, \hbox {NPs}\) was found to be 0.433, 23.498 and \(8.897 \, \hbox {m}^{2} \, \hbox {g}^{-1}\) respectively and the total pore volume was 0.002, 0.081 and \(0.021 \, \hbox {cc g}^{-1}\) respectively (table 2). The surface area played an important role in the observed trend of activity. More the surface area of ferrite NPs, more the sites available for substrate attachment and hence higher was the activity.

3.2 Evaluation of SOD mimic activity of \({\hbox {MFe}}_{2} \hbox {O}_{4}\)

3.2.1 Effect of pH

For evaluating the effect of pH on SOD mimic activity, the pH of the reaction mixture was varied from 3.0–10.0 using 0.1 N HCl and 0.1 N NaOH and absorbance was noted after 10 min. It was observed that the activity of ferrite NPs first decreased on increasing the pH up to 8.0 (figure 5(a)) as indicated by higher absorbance values of the solution and subsequently increase which was affirmed by lowering in absorbance value at pH > 8. The results might be due to interaction of H\(^+\) ions with \(\text {O}_2^-\) which inhibits its binding with metal ion containing SOD / SOD mimic.

Table 2 BET surface area of ferrite NPs.
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Fig. 5
figure 5

(a) Effect of pH on SOD mimic activity of \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs, (b) comparison of SOD mimic activity of ferrite NPs, (c) effect of contact time on SOD mimic activity of \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs and (d) effect of \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NP dose on SOD mimic activity.

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Fig. 6
figure 6

Steady-state kinetic assay of SOD mimic activity of \({\hbox {CuFe}}_{2} {\hbox {O}}_{4}\) NPs.

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3.2.2 Comparison of SOD mimic activity of ferrite NPs

The addition of \(\hbox {CuFe}_{2} \hbox {O}_{4} \hbox { NPs}\) resulted in a maximum lowering in absorbance of the reaction mixture followed by \(\hbox {CoFe}_{2} \hbox {O}_{4}\) and \(\hbox {MnFe}_{2} \hbox {O}_{4} \hbox { NPs}\). The results were correlated with the inter-conversion of the oxidation state during superoxide dismutation. The highest activity of \(\hbox {CuFe}_{2} \hbox {O}_{4} \hbox { NPs}\) can be attributed to the facile conversion of \(\hbox {Cu}^{2+}\) to \(\hbox {Cu}^{+}\) due to the electron transfer from a substrate containing superoxide radical to \(\hbox {CuFe}_{2} \hbox {O}_{4} \hbox { NPs}\) that lead to more stable \({\hbox {3d}}^{10}\) configurations from \({\hbox {3d}}^{9}\) configurations. Whereas, in the case of \(\hbox {CoFe}_{2} \hbox {O}_{4}\) and \(\hbox {MnFe}_{2} \hbox {O}_{4} \hbox { NPs}\) the change in the oxidation state from +2 to +1 did not lead to a favourable electronic configuration. A similar mechanism is reported in the case of the Cu–Zn SOD enzyme during dismutation of the \(\hbox {O}_{2}^{-}\) anion [25]. The higher activity of \(\hbox {CoFe}_{2} \hbox {O}_{4}\) as compared to \(\hbox {MnFe}_{2} \hbox {O}_{4} \hbox { NPs}\) might be explained on the basis of the higher surface area of the former which resulted in more available reactive sites for the attachment of substrates and thus scavenging the superoxide radicals at a faster rate (figure 5(b)).

3.2.3 Effect of contact time and catalyst dose of ferrite NPs

To observe the effect of contact time, SOD mimic activity was evaluated for 10 min at an interval of 30 s. The results showed that the maximum activity was achieved within 3 min, afterwards, no significant change in activity was observed. Thus, for subsequent experiments the value of absorbance was noted after 3 min (figure 5(c)).

Scheme 1
scheme 1

Proposed mechanism of SOD/SOD mimic activity.

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In the reaction mixture, ferrite NPs (0.5–5 mg) were added. The activity first increased on addition of ferrite NPs up to 2 mg followed by a non-significant change in absorbance up to 5 mg (figure 5(d)). The increase in activity on increasing dose of NPs was due to availability of more active sites, but at an optimum dose of 2 mg the substrate was saturated on the active sites of NPs and further increase in the dose of NPs did not result in any increase in activity. Thus, 2 mg of NPs for 3 ml reaction solution (\(0.66 \, {\hbox {mg ml}}^{-1}\)) was the optimum enzyme mimic dose.

3.2.4 Steady-state kinetics

To investigate the catalytic mechanism of \(\hbox {CuFe}_{2} \hbox {O}_{4} \hbox { NPs}\), apparent steady-state kinetic parameters for the SOD mimic reaction were measured using pyrogallol as the substrate. The parameters were obtained by varying the substrate concentrations. The slope and intercept of the graph gave the value of the maximum velocity (\(v_{\mathrm{max}}\)) and Michaelis Menten constant (\(K_{\mathrm{m}}\)) (figure 6). The \(v_{\mathrm{max}}\) and \(K_{\mathrm{m}}\) obtained were \(0.77 \, \hbox {s}^{-1}\) and 4.20 mM respectively which were comparable to the reported value for SOD enzyme values in the literature [26]. The optimum pH for naturally available SOD is 9, whereas ferrite nanoparticles showed a maximum SOD like activity at pH 1. Thus the direct comparison was not undertaken as SOD and ferrite NPs did not show a maximum activity under identical conditions of pH due to difference in their structural features. Therefore activity was compared with the reported values for SOD in the literature. The proposed catalytic mechanism is shown in scheme 1. The superoxide anion first binds to \(\hbox {Cu}^{2+}\) of ferrite NPs and reduces \({\hbox {Cu}}^{2+}\) to \({\hbox {Cu}}^{+}\). Further protonation resulted in the loss of dioxygen and the conversion of \({\hbox {Cu}}^{+}\) back to \({\hbox {Cu}}^{2+}\).

4 Conclusions

In the present study, the effect of a metal ion in spinel ferrite NPs on SOD mimic activity was observed. A facile sol–gel method was used to synthesize \(\hbox {MFe}_{2} \hbox {O}_{4} \hbox { NPs}\) (\(\hbox {M} = \hbox {Mn}\), Co and Cu). Although, all the synthesized ferrite NPs showed SOD mimic activity, the ferrite NPs containing \({\hbox {Cu}}^{2+}\) as a divalent ion showed a maximum SOD mimic activity followed by \(\hbox {CoFe}_{2} \hbox {O}_{4}\) and \(\hbox {MnFe}_{2} \hbox {O}_{4} \hbox { NPs}\). The observed trend was attributed to the facile conversion of \({\hbox {Cu}}^{2+}\) to \({\hbox {Cu}}^{+}\) that led to a more stable configuration. The higher activity of \(\hbox {CoFe}_{2} \hbox {O}_{4}\) as compared to \(\hbox {MnFe}_{2} \hbox {O}_{4} \hbox { NPs}\) was explained on the basis of the higher surface area of the former that provided more reactive sites. Kinetic analysis of \(\hbox {CuFe}_{2} \hbox {O}_{4} \hbox { NPs}\) confirmed the potential of \(\hbox {CuFe}_{2} \hbox {O}_{4}\) as a SOD mimic.