Enhancement of peroxidase-like activity of N-doped graphene assembled with iron-tetrapyridylporphyrin

Abstract

The paper describes two preparation methods for the non-covalent functionalization of N-doped graphene with iron(III)-tetrapyridylporphyrin. The obtained nanomaterials show enhanced peroxidase-like activity compared to N-doped graphene and effectively oxidize 3,3′-dimethylbenzidine with hydrogen peroxide with the formation of a blue product. The catalytic activity and implicitly the color intensity are dependent on the hydrogen peroxide concentration, thus creating the possibility for its quantification by UV-Vis spectroscopy. In addition, the nanomaterial obtained by direct functionalization was investigated for its catalytic ability to remove bisphenol A from pH neutral water solutions.

---

1. Introduction

Currently, carbon nanostructures represent a widely explored class of nanomaterials, due to the unique characteristic, physicochemical and biological properties which have inspired their vast utility. Expanding beyond nanotechnology towards biology, carbon nanomaterials have demonstrated their applicability also in biomimetics. Even though it might seem less productive to replace the enzymes' high substrate specificity and yields, in some cases nanomaterials overcame their limitations regarding low stability, high sensitivity to environmental conditions and high production costs.¹ Over the past decade, carbon nanomaterials have been investigated as SOD-like,^2,3 oxidase-like⁴ and, particularly interesting for us, as peroxidase-like nanomaterials.^5–7 Graphene-based nanomaterials are among the most studied nanomaterials of the last decade. As far as their peroxidase-like activity is involved, we recently showed that their intrinsic catalytic activity towards dimethylbenzidine oxidation in the presence of hydrogen peroxide is dependent on the graphene structure.⁸ The biomimetic behaviour was either due to the known catalytic activity of supported metallic nanoparticles (e.g. gold or platinum) or to the functional groups at the surface of graphenes. The nitrogen-doped graphene showed a remarkable catalytic activity due to the nitrogen atoms inserted into the carbon lattice and proved a good metal-free candidate support for developing new nanocomposites for various applications.⁸

Porphyrins and metalloporphyrins are well known macrocyclic derivatives, found as components in many biological systems, especially enzymes including myoglobins, hemoglobins, cytochrome C oxidase, vitamin B12, chlorophyll and peroxidases.⁹ As a consequence, porphyrins have been employed for the preparation of various nanocomposites with applicability in biomimetic catalysis.^10–13 In a previous report, we showed that both graphene oxide and thermally reduced graphene oxide present improved peroxidase-like activity when iron-tetrapyridylporphyrin was non-covalently assembled on their surface,¹⁴ but the catalytic properties of the functionalized N-doped graphene have not been yet explored.

As such, the present study is describing the characterization of tetrapyridylporphyrin and iron-tetrapyridylporphyrin assembled on N-doped graphene by non-covalent interactions. The N-doped graphene–porphyrin composites were obtained by two routes and were evaluated for their peroxidase-like activity towards the oxidation of dimethylbenzidine in the presence of hydrogen peroxide. So far, graphene-based composites with peroxidase-like activity have been mainly employed in the field of sensors.

It is known that the catalytic properties of peroxidases were highlighted in phenols and aromatic amines removal. The enzymatic degradation of bisphenol A at micromolar range concentrations has been conducted with either horseradish peroxidase¹⁵ or laccase immobilized on carbon nanomaterials.¹⁶ The application of peroxidase-like activity of nanozymes for pollutant removal has been previously applied for phenol degradation.¹⁷ In this respect, we implemented the improved activity of the iron-porphyrin functionalized N-doped graphene for the catalytic removal of bisphenol A.

2. Materials and methods

All the reagents were of analytical grade and used without further purification. 3,3′-Dimethylbenzidine (ortho-tolidine) and 5,10,15,20-tetra(4-pyridyl)porphyrin (TPyP) were purchased from Sigma-Aldrich. Dimethylsulfoxide (DMSO) and urea were purchased from Merck. N-doped graphene and iron(III)tetrapyridylporphyrin were prepared according to a previously described synthesis.^8,14 The water was purified with a Milli-Q system (Millipore, USA) and acetonitrile (HPLC Gradient Grade) was purchased from LGC standards (GmbH, Germany).

The morphology of graphene nanocomposites was observed by transmission electron microscopy (TEM) (H-7650 120 kV Automatic TEM, Hitachi, Japan) and high-resolution transmission electron microscopy (SU-8230 STEM – Hitachi, Japan). For TEM images, the samples diluted in ethanol were dropped on a copper grid. The ultraviolet-visible (UV-Vis) absorption spectra of enzyme mimic and the time-dependent absorbance spectra were measured with a V-570, JASCO Spectrophotometer. X-ray Photoelectron Spectroscopy (XPS) technique has been used for the characterization of chemical composition and state of elements present in the investigated graphenes. XPS spectra were recorded using a SPECS spectrometer, equipped with a dual-anode X-ray source Al/Mg, a PHOIBOS 150 2DCCD hemispherical energy analyzer and a multi-channeltron detector. The pressure inside the measurement chamber was maintained constant at about 1 × 10⁻⁹ torr. The sample, as a colloidal suspension in methanol, was dried in successive layers on indium foil stacked on wolfram sample holder. Irradiation was made with an Al_Kα X-ray source (1486.6 eV) operated at 200 W. The XPS survey spectra were recorded at 30 eV pass energy, 0.5 eV per step. The high resolution spectra for individual elements were recorded by accumulating 10–15 scans at 30 eV pass energy and 0.1 eV per step. The surface cleaning was ensured through argon ion bombardment at 300 V for 2, 3 and 5 minutes. Data analysis and experimental curve fitting of the C 1s, N 1s and Au 4f spectra was performed using Casa XPS software with a Gaussian–Lorentzian product function and a non-linear Shirley background correction. The Raman spectra were collected at room temperature, using a JASCO type NRS3300 spectrophotometer arranged in a backscattering geometry, coupled to a CCD (−69 °C) detector with a 1200 l per mm grid and a spectral resolution of 7.5 cm⁻¹. The incident laser beam with a 1 μm² diameter was focused through an Olympus microscope (100× objective), and the calibration was made using the Si peak at 521 cm⁻¹. The excitation was done using an Ar-ion laser with a wavelength of 514 nm and a power at the sample surface of 1.1 mW.

2.1 Preparation of N-doped graphene–porphyrin nanocomposites

A suspension of N-doped graphene (35 mg) in 20 mL ethanol was sonicated for 30 min. A volume of 350 μL of TPyP or Fe(III)TPyP (5 mM in DMF) was added and the final mixture was stirred at room temperature, overnight. The dispersion was then centrifuged (3000 rpm for 25 min) and the supernatant discarded. The remaining solid was washed three more times with the same volume of ethanol, followed by centrifugation. Finally, the solid was dried at 35 °C. The resulting nanocomposites were following denoted NGr-(Fe)TPyP.

In the other procedure, a suspension of N-doped graphene intermediate (50 mg) in 20 mL ethanol was sonicated for 30 min. In each solution, a volume of 350 μL of TPyP or Fe(III)TPyP (5 mM in DMF) was added and the solution was stirred at room temperature, overnight. The final dispersion was then centrifuged (3000 rpm for 25 min) and the supernatant discarded. The solid was washed three more times with the same volume of ethanol, followed by centrifugation. Finally, the solid was treated at 500 °C in the oven, in argon flow. The resulting nanocomposites were following denoted (Fe)TPyP-NGr.

2.2 Peroxidase catalytic activity experiments

The catalytic activity study of all graphene-based nanomaterials was performed as follows: 230 μL of graphene suspension (2.5 mg in 5 mL of 0.5% HCl) was added in a reaction volume of 3 mL acetate buffer solution (ABS, 0.2 M, pH 4.2) with H₂O₂ (1 mM) and ortho-tolidine (1 mM).

The kinetic analysis were carried out with 230 μL of graphene suspension (2.5 mg in 5 mL of 0.5% HCl) in a reaction volume of 3 mL ABS (0.2 M, pH 4.2) with ortho-tolidine (0.8 mM final concentration) and variable H₂O₂ concentration. The reactions were carried out in a quartz cuvette with an optical path length of 1 cm and the time course measurement was set at 630 nm.

2.3 Experiments for bisphenol A

The reactions were performed at room temperature in a total volume of 3 mL phosphate buffer (pH 7.0) containing predetermined amounts of bisphenol A, a suspension of NGr-FeTPyP and H₂O₂. Hydrogen peroxide was added as the final reagent to initiate the catalytic reaction and the reaction mixture was let for stirring. At each time interval, the reaction mixture was filtered to separate the solids, and the liquid phase was taken for HPLC analysis. Liquid chromatographic analyses of bisphenol A were performed using an ultra high liquid chromatography system (Accela UHPLC system, Thermo Scientific, USA) equipped with quaternary pump, autosampler and UV-Vis detector with a photodiode array (PDA). Chromatographic separations were carried out on a BDS Hypersil C18 column (150 mm × 4.6 mm, 5 μm particle size) under an isocratic program using 35% of water (solvent A) and 65% of acetonitrile (solvent B) as mobile phase components. The flow rate was set at 0.5 mL min⁻¹, the injection volume was 5 μL and detection was monitored at λ = 229 nm. The bisphenol A experiments have been performed over a period of three months with the same catalyst suspension without changes in the catalytic activity. In its solid form the catalyst was kept for over one year.

3. Results and discussion

3.1 Preparation and morphological characterization of composites

This work uses tetrapyridylporphyrin and its iron(III)complex for the non-covalent functionalization of N-doped graphene, thus obtaining highly efficient biomimetic catalysts. The nitrogen atoms in the graphene lattice offer plenty of binding sites for non-covalent functionalization of iron-porphyrin through Fe–N coordination,¹⁸ while the free porphyrin bind solely through the π–π interactions between the aromatic rings of the porphyrin and the aromatic carbon islands of the graphene. The synthesis of N-doped graphene is a two step procedure: introduction of the nitrogen containing functional groups by the reaction of graphene oxide with urea, followed by a thermal treatment (600 °C) that lead to re-annealing of the aromatic structure and insertion of the pyridinic, pyrrolic or graphitic nitrogen atoms into the lattice. Therefore, we found of interest to investigate the effect of functionalization with (iron)porphyrin either in the intermediate step (NGr-intermediate), followed by the necessary thermal treatment (Ar, 500 °C) with the formation of (Fe)TPyP-NGr, or direct functionalization of the already annealed N-Gr thus obtaining NGr-(Fe)TPyP (Fig. 1). The temperature for the treatment of the intermediate containing (iron)porphyrin was set lower (500 °C vs. 600 °C) in order to avoid the degradation of the organic molecule.

Fig. 1 Preparation of porphyrin functionalized N-doped graphenes.

Further, the physical and chemical properties of these nanocomposites with porphyrins were evaluated in order to conclude on the most effective route for functionalization of N-doped graphene with these macromolecules.

The morphological characterization shows similar structures for the four nanocomposites. The TEM images display folded graphene sheets with typical morphology as a result of the thermodynamic stability of the conjugated system.¹⁹ The functionalization with porphyrins is not causing important changes of the graphene flakes structure (Fig. 2). All four composites present porphyrin aggregates of different sizes. In order to observe their structure, we performed HR-TEM characterization of the samples. We obtained nice images of the graphene layers for all samples (Fig. S1†), but the porphyrin aggregates are embedded in areas of graphene with more layers and we were unable to obtain their high resolution lattice.

Fig. 2 TEM images of porphyrin functionalized N-doped graphenes.

X-ray diffraction patterns for the TPyP- and FeTPyP-containing N-doped graphene are shown in Fig. 3(a) and (b), compared with the patterns for the starting N-doped graphene (Fig. 3(a) and (b), black lines) and the free porphyrin (Fig. 3(a) and (b), brown lines). All nanocomposites present the specific peak of graphenes at θ = 25° split up into two components. The appearance of two distinct maxima with slightly different interplanar spacing can be explained by the uneven distribution of the nitrogen atoms in the carbon lattice.

Fig. 3 XRD patterns of the porphyrin functionalized N-doped graphenes.

Because the diffraction peaks corresponding to the two maxima of graphenes are partly overlapped, the crystallites size was measured after deconvolution. These crystallites are perpendicular to the (002) graphene plane. The interplanar distances corresponding to each peak as well as the number of layers were also determined (Table S1, ESI†). The main observation is that the crystallites size for the nanocomposites prepared by the direct method (NGr-TPyP and NGr-FeTPyP) is generally larger than the one of the graphenes obtained starting from the intermediate NGr (TPyP-NGr and FeTPyP-NGr).

In addition, the porphyrin-functionalized N-doped graphenes obtained by the direct method (NGr-TPyP and NGr-FeTPyP) show also the most intense peaks corresponding to the free porphyrin, at 19.8° and 21.2° (Fig. 3(a) and (b)). In the nanomaterials obtained starting from the intermediate NGr (TPyP-NGr and FeTPyP-NGr) the corresponding peaks have much lower intensities corresponding to a less efficient functionalization. This fact corroborate well with the above spectroscopic results that conclude on a less efficient material, when starting from the intermediate of NGr.

3.2 Spectroscopic characterization

The porphyrin-N-doped graphene hybrid nanosheets were characterized by various spectroscopic methods. UV-Vis spectra of the NGr nanocomposites with TPyP and FeTPyP and the corresponding porphyrins are depicted in Fig. 4(a) and (b). Both TPyP and FeTPyP exhibit the typical Soret-band absorption at λ = 413–414 nm. In the presence of N-doped graphene the Soret-band present a decrease in the intensity for the nanocomposites (Fe)TPyP-NGr obtained from the intermediate of the nitrogen doping, indicating a lower amount of porphyrins present on the surface. This effect is dramatic in the case of iron-complexed porphyrin (Fig. 4(b), blue line). An interesting observation is the Soret-band lack of shifting in the presence of N-doped graphene compared to our data on graphene oxide and thermally reduced graphene oxide.¹⁴ The different behavior is probably caused by the modifications of the electronic interaction of porphyrins with graphene, induced by the presence of nitrogen atoms in the graphene network.²⁰

Fig. 4 UV-Vis spectra of the porphyrin functionalized N-doped graphenes.

The Raman spectra for (Fe)TPyP-NGr and NGr-(Fe)TPyP are represented in Fig. 5(a) and (b), alongside the spectrum of N-doped graphene (N-Gr, green lines) and tetrapyridylporphyrin (TPyP, brown lines). As a general remark, the spectrum of NGr exhibits the two characteristic Raman bands for graphene,²¹ the D band at 1357 cm⁻¹, slightly down-shifted compared to the thermally reduced graphene oxide (1360 cm⁻¹)¹⁴ due to the presence of nitrogen atoms in the sp² network. The D band is generally associated with the structural defects of the graphene, and its intensity is growing with the number of layers.²² The G band is characteristic to all sp² carbon nanomaterials and appears at 1599 cm⁻¹, approximately 5 cm⁻¹ up-shifted compared to the reduced graphene.¹⁴ The structure modifications induced by the insertion of nitrogen sites within the carbon network caused the above mentioned Raman bands shifts.

Fig. 5 Raman spectra of the porphyrin functionalized N-doped graphenes.

The Raman spectra of the nanocomposites with (Fe)TPyP (red and black lines, Fig. 5) present the same main bands together with shoulders and even well-defined peaks corresponding to the Raman bands of porphyrin, for example at 324, 1005, 1250, 1460, 1559 cm⁻¹. They are more visible for the nanocomposites obtained by direct functionalization from NGr (NGr-TPyP and NGr-FeTPyP, black curves) than the ones prepared starting from NGr intermediate (TPyP-NGr and FeTPyP-NGr, red curves). This indicates that the non-covalent functionalization of N-doped graphene with the two porphyrins was more efficient by the direct method. This conclusion correlate well with the low intensity of the Soret-band absorption for the nanocomposites prepared from the NGr intermediate ((Fe)TPyP-NGr), suggesting that a low amount of porphyrins was adsorbed on the graphene surface. Additionally, the analysis of the Raman spectra reveal that the G band is down-shifted with 3 cm⁻¹ when TPyP is attached to NGr and the shift is even larger for FeTPyP (9 cm⁻¹). As such, the iron containing porphyrin is strongly interacting with the N-doped graphene, probably by Fe–N coordination. The ratio I_D/I_G is apparently unchanged for all porphyrin-NGr nanocomposites compared to NGr (approx. 1.0), showing that the functionalization is not affecting the conjugation of the aromatic system.²³

X-ray photoelectron spectroscopy spectra of NGr-FeTPyP and FeTPyP-NGr nanocomposites were measured in order to obtain more informations on the chemical functionalities. The C 1s high resolution spectra of NGr-FeTPyP (obtained by direct functionalization of the already annealed N-Gr with iron porphyrin) and that of FeTPyP-NGr (resulted from the functionalization with iron porphyrin in the intermediate step followed by thermal treatment) are shown in Fig. 6. In both cases the deconvolution of the experimental spectrum was made with six different components with a Gaussian–Lorentzian shape. The spectra approximation is dominated by a very intense peak attributed to the carbon sp² hybridized atoms (both surface and bulk), located at binding energies (BE) of 284.27 eV and 284.31 eV, respectively. Depending on the different chemical environment experienced by the carbon atoms, the C 1s high resolution spectra also highlight the significant variations in carbon species present on the graphene surface. At the edges of the sp² network, structural disorder is found: carbon–hydrogen bonded groups in a sp³ hybridized state which appear at 284.88 eV and 284.86 eV respectively. In addition, carbon–oxygen (C–O–C, C=O, O=C–O and COOH)²⁴ and carbon–nitrogen (C–N)²⁵ bonded groups are also present (see Fig. 6 and Table S2, ESI†). In the high binding energy region (292.23 eV and 291.83 eV) a small contribution assigned to the π → π* shake-up satellite band of graphitic carbons appears.⁸

Fig. 6 Core level high-resolution C 1s XPS spectra of NGr-FeTPyP (a) and that corresponding to FeTPyP-NGr (b).

The incorporation of nitrogen in the carbon framework is confirmed by the photoemission from the N 1s levels. In case of both samples more nitrogen species are present, thus the deconvolution of the asymmetric N 1s high resolution spectrum needed three characteristic peaks corresponding to: pyridinic (referring to the nitrogen atoms bonded to two carbon nearest neighbors at the edge of graphene planes that is capable of adsorbing molecular oxygen), pyrrolic (represents the nitrogen atoms that contribute to the π system with two p-electrons) and graphitic nitrogen (which is sometimes termed “quaternary” nitrogen and represents the nitrogen atoms bonded to three carbon atoms within a graphite basal plane). The position of these peaks is comparable in both samples, but the quantification of their contributions is slightly different (see Fig. 7 and Table S2, ESI†). It should be noted that the NGr-FeTPyP sample has larger fraction of the pyridinic nitrogen groups compared to the pyrrolic and graphitic ones and it shows higher catalytic activity compared to FeTPyP-NGr, indicating that the pyridinic nitrogen group is active for catalytic activity. Thus, the higher catalytic activity observed in case of NGr-FeTPyP sample is also due to the increased number of active sites. The C/N ratio was found out to be 7.96 for NGr-FeTPyP and 4.10 for FeTPyP-NGr respectively.

Fig. 7 High-resolution XPS N 1s core level spectra corresponding to NGr-FeTPyP (a) and FeTPyP-NGr (b).

The structural defects and the presence of different functional groups at the surface of investigated materials are confirmed in the O 1s high resolution spectrum (see Fig. 8 and Table S2, ESI†). For both samples the oxygen atoms are bonded with the unsaturated carbon atoms present at graphene edge sites in the form of C–O, C=O and O–C=O oxygen-containing groups. At high binding energy the presence of a small contribution of 13.86% for NGr-FeTPyP and 9.83% for FeTPyP-NGr respectively, coming from the adsorbed water molecules could be observed. In case of NGr-FeTPyP the functionalization of the N-doped graphene nanosheets surface with iron porphyrin is confirmed by the presence in the low binding energy region of the Fe–O contribution, centered at about 529.64 eV. For FeTPyP-NGr sample, the quantity of iron-porphyrin on the surface of N-doped graphene is smaller and its presence could not be detected by XPS measurement, as observed in the UV and Raman spectra. The C/O ratio was determined to be 1.22 in case of NGr-FeTPyP and 1.24 for FeTPyP-NGr respectively.

Fig. 8 High-resolution XPS O 1s core level spectra corresponding to NGr-FeTPyP (a) and FeTPyP-NGr (b).

The spin–orbit coupling gives rise to a doublet of lines in the Fe 2p high resolution XPS spectrum (Fe 2p_3/2 – 711.75 eV and Fe 2p_1/2 – 724.51 eV) of NGr-(Fe)TPyP sample, confirm the successful functionalization of N-doped graphene surface with iron-porphyrin (Fig. 9); but the low spectrum resolution, due to the small quantity made the fitting impossible. In case of FeTPyP-NGr the photoemission from Fe 2p core levels is not visible. As such, we performed also the element mapping using analytical electron microscopy techniques and clearly saw that iron existed in the sample, but in small quantities (Fig. S2a†). The presence of iron element was also confirmed by the EDX spectrum of the sample (Fig. S2b†).

Fig. 9 High-resolution XPS Fe 2p core level spectra corresponding to NGr-FeTPyP and FeTPyP-NGr.

3.3 Peroxidase-like activity and its applications

The peroxidase-like activity of the prepared NGr-(Fe)TPyP nanocomposites was measured in sodium acetate buffer by oxidizing the substrate (3,3′-dimethylbenzidine) in the presence of hydrogen peroxide. The reaction is monitored by the formation of a blue colored product, corresponding to the one-electron oxidation of 3,3′-dimethylbenzidine, in a rapid equilibrium with the charge-transfer complex of the aromatic diamine (Fig. 10). The maximum absorbance wavelength for the primary oxidation product is 630 nm.²⁶ No color development was observed in the absence of any N-doped graphene-based nanomaterial, indicating their catalytic ability in this reaction.

Fig. 10 Reaction scheme for peroxidase-like activity.

As stated in the introduction, one of the goals of the present work was to evaluate the improvement of the catalytic activity of the N-doped graphene by the functionalization with tetrapyridylporphyrin and its iron(III) complex. Therefore, the previously optimized experimental conditions were employed also for measuring the actual nanomaterials. Namely, the optimum graphene concentration of 40.6 μg mL⁻¹ was mixed with a solution of 3,3′-dimethylbenzidine in acetate buffer (pH = 4.2) at room temperature and the color development was followed for 20 minutes after the addition of hydrogen peroxide. The time dependent absorbance changes measured at 630 nm are represented in Fig. 11.

Fig. 11 The time-dependent absorbance changes of NGr-based porphyrin nanomaterials in pH 4.2 acetate buffer at room temperature.

The changes in absorbance are observed for all the solutions, indicating that the peroxidase-like activity of the different nanomaterials increases with time. The comparison between the absorbance intensities clearly show a quicker increase in case of the iron-porphyrin functionalized N-doped graphenes (NGr-FeTPyP and FeTPyP-NGr)/DMB/hydrogen peroxide systems than for the systems formed with the N-doped graphenes and uncomplexed porphyrin (NGr-TPyP and TPyP-NGr). In the same time, these systems containing TPyP show very little difference in the time variation of the absorbance at 630 nm compared to the parent N-doped graphene or even the unsupported iron-porphyrin. The lower catalytic activity of the free iron-porphyrin is due to the formation of catalytically inactive dimers.²⁷

These observations indicate a significant higher catalytic activity of the iron-porphyrin functionalized N-doped graphene catalysts. Their higher peroxidase-like activity is due to the cumulative effect exerted by the presence of different type of nitrogen atoms on one hand, and of the iron-porphyrin on the surface of N-doped graphene on the other hand. The catalytic mechanism of nitrogen atoms was not completely elucidated yet, though it is known that the pyrrolic, pyridinic and graphitic nitrogen atoms play different roles.⁸ The iron-porphyrin is known as more catalytically active when functionalized on graphenes because it can keep its active monomeric form, the self-aggregation being hindered.

In order to further study the peroxidase-like activity of the two FeTPyP containing N-doped graphenes, we determined the apparent steady-state kinetic on the oxidation of 3,3′-dimethylbenzidine in the presence of H₂O₂ and FeTPyP-NGr or NGr-FeTPyP as catalysts. We applied the enzyme kinetics using hydrogen peroxide as substrate, therefore, the experiments were performed by changing H₂O₂ concentration while keeping all the other constant (Fig. 12(a) and (b)).

Fig. 12 Time-dependent absorbance changes at 630 nm at different concentrations of H₂O₂ for NGr-FeTPyP (a) and FeTPyP-NGr (b).

The typical Michaelis–Menten curves for H₂O₂ were obtained by monitoring the absorbance at 630 nm for 10 minutes (Fig. 13(a) and (b)). The initial reaction rate is linear for both iron-porphyrin containing N-doped graphene thus indicating a first order reaction. The double reciprocal plots between the reaction rates and hydrogen peroxide concentrations show a good linearity, further indicating that the oxidation reaction of dimethylbenzidine catalyzed by N-doped graphene modified with iron-porphyrin follows a Michaelis–Menten kinetics.

Fig. 13 Steady-state kinetic assay of NGr-FeTPyP (a), FeTPyP-NGr (b) and the corresponding Lineweaver–Burk plots (insets). Experiments were carried out in acetate buffer (0.2 M; pH 4.2) with a concentration of 0.8 mM for ortho-tolidine.

The kinetic parameters using H₂O₂ as substrate are K_m = 0.261 mM and v_max = 7.18 × 10⁻⁴ s⁻¹ for the reaction with NGr-FeTPyP, while for the reaction with FeTPyP-NGr are K_m = 0.515 mM and v_max = 9.08 × 10⁻⁴ s⁻¹. A low K_m value indicates a better affinity of the nanomaterial towards hydrogen peroxide. By comparing these values with previous data (Table 1), the affinity constant of NGr-FeTPyP with hydrogen peroxide as substrate is much smaller than that of horseradish peroxidase measured under the same experimental conditions. The K_m value is also smaller than that of unfunctionalized NGr supporting the observed behavior in the time dependent absorbance changes experiment (Fig. 11). For FeTPyP-NGr catalyst the K_m value with hydrogen peroxide as substrate is higher than that of NGr, even though the time dependent absorbance changes at 630 nm show a better catalytic activity.

Table 1 Comparison of the kinetic parameters of various nanomaterials

Nanomaterial	Km (mM)-H2O2 as substrate	Experimental conditions	Ref.
a Measured with dimethylbenzidine.b Measured with tetramethylbenzidine.
NGr-FeTPyP^a	0.261	Room temperature, 0.2 M acetate buffer pH 4.2	This work
FeTPyP-NGr^a	0.515	Room temperature, 0.2 M acetate buffer pH 4.2	This work
Hemin^b	2.74	Room temperature, 0.025 M phosphate buffer pH 5	10
Ch. reduced GO–hemin^b	2.256	Room temperature, 0.025 M phosphate buffer pH 5	10
N-doped graphene^a	0.486	Room temperature, 0.2 M acetate buffer pH 4.2	8
HRP^b	3.70	Temperature: 40 °C, 0.2 M acetate buffer pH 3.5	28
	0.214	Temperature: 35 °C, 0.025 M phosphate buffer pH 4	7
HRP^a	1.579	Room temperature, 0.2 M acetate buffer pH 4.2	8

---

The catalytic properties of nanozymes have been mainly utilized for development of sensors that take advantage of the color intensity dependence on the hydrogen peroxide concentration.²⁹ For the colorimetric detection using the NGr-FeTPyP nanocomposite, the experimental data show a linear range within 1–160 × 10⁻⁶ M (Fig. 14). The absorbance values were determined from the time course measurements and correspond to the 10 min reaction with the NGr-FeTPyP/o-tolidine system. The limit of detection was calculated at 5.6 × 10⁻⁶ M, indicating a better catalytic activity than previously reported for GO-FeTPyP nanocomposite.

Fig. 14 Absorbance at 630 nm vs. H₂O₂ concentration for the NGr-FeTPyP nanocomposite. The inset shows the photos of the solutions with different concentrations of H₂O₂ (from left to right: 0, 0.001, 0.5, 2 mM).

Further, we were also interested in another application for the improved peroxidase-like activity of NGr-FeTPyP: the removal of bisphenol A from water solutions. The experiments for testing the bisphenol A degradation were carried out in phosphate buffer solution (pH = 7), at room temperature. The catalyst concentration was set at 0.05 g L⁻¹ of NGr-FeTPyP, while for the initial bisphenol A two concentrations have been considered: 10 mg L⁻¹ vs. 34.3 mg L⁻¹. The investigation of a lower concentration is necessary since it was stated previously that endocrine disrupting materials are biologically active even at very low concentrations.³⁰ The percentage of residual bisphenol A was determined with HPLC, by integrating the area of the peak with the retention time at 4.47 min. As shown in Fig. 15, 58% of the bisphenol A is degraded in 24 hours, in the presence of 100 mM H₂O₂ (black line). The removal efficiency increased up to 68% when the concentration of hydrogen peroxide was lowered at 10 mM. An enhanced degradation when decreasing the hydrogen peroxide concentration has been also observed for the enzymatic degradation of bisphenol A.³¹ The degradation process became very slow when the starting bisphenol A concentration was set at 10 mg L⁻¹ (blue line). These data indicate the potential of NGr-FeTPyP to act as an inexpensive catalyst for the removal of bisphenol A, avoiding the disadvantages employed by the enzymatic methods.

Fig. 15 Removal efficiency of bisphenol A in the presence of 50 mg L⁻¹ NGr-FeTPyP ((A) 34.3 mg L⁻¹ bisphenol A, 100 mM H₂O₂; (B) 34.3 mg L⁻¹ bisphenol A, 10 mM H₂O₂; (C) 10 mg L⁻¹ bisphenol A, 10 mM H₂O₂).

4. Conclusions

We synthesized iron-porphyrins containing N-doped graphenes by employing two preparation procedures. The physico-chemical characterization of the nanomaterials led to the conclusion that functionalizing the N-doped graphene after thermal treatment is more efficient, as more porphyrin has been non-covalently attached to the surface. The NGr-FeTPyP showed an improved peroxidase-like catalytic activity that was applied to develop a sensitive colorimetric method for hydrogen peroxide. In addition, the unique catalytic activity found application in the efficient degradation of bisphenol A (up to 80%), further increasing the potential of nanomaterials with peroxidase-like activity towards wastewater treatment.

Acknowledgements

This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, Project Number PN-II-RU-TE-2014-4-0305. The authors thank Mr Sebastian Porav for his help with the HR-TEM measurements.

References

1. H. Wei and E. Wang, Chem. Soc. Rev., 2013, 42, 6060–6093.
2. S. S. Ali, J. I. Hardt, K. L. Quick, J. S. Kim-Han, B. F. Erlanger, T. T. Huang, C. J. Epstein and L. L. Dugan, Free Radical Biol. Med., 2004, 37, 1191–1202.
3. K. L. Quick, S. S. Ali, R. Arch, C. Xiong, D. Wozniak and L. L. Dugan, Neurobiol. Aging, 2008, 29, 117–128.
4. B. Zhang, Y. He, B. Liu and D. Tang, Anal. Chim. Acta, 2014, 851, 49–56.
5. B. Garg, T. Bisht and Y. C. Ling, Molecules, 2015, 20, 14155–14190.
6. Y. Song, X. Wang, C. Zhao, K. Qu, J. Ren and X. Qu, Chem.–Eur. J., 2010, 16, 3617–3621.
7. Y. Song, Z. Qu, C. Zhao, J. Ren and X. Qu, Adv. Mater., 2010, 22, 2206–2210.
8. F. Pogacean, C. Socaci, S. Pruneanu, A. R. Biris, M. Coros, L. Magerusan, G. Katona, R. Turcu and G. Borodi, Sens. Actuators, B, 2015, 213, 474–483.
9. S. Shaik, S. Cohen, Y. Wang, H. Chen, D. Kumar and W. Thiel, Chem. Rev., 2010, 110, 949–1017.
10. T. Guo, L. Deng, J. Li, S. Guo, E. Wang and S. Dong, ACS Nano, 2011, 5, 1282–1290.
11. Y. Xu, L. Zhao, H. Bai, W. Hong, C. Li and G. Shi, J. Am. Chem. Soc., 2009, 131, 13490–13497.
12. Q. Liu, Y. Yang, H. Li, R. Zhu, Q. Shao, S. Yang and J. Xu, Biosens. Bioelectron., 2015, 64, 147–153.
13. Q. Liu, H. Li, Q. Zhao, Y. Yang, Q. Jia, B. Bian and L. Zhuo, Mater. Sci. Eng., C, 2014, 41, 142–151.
14. C. Socaci, F. Pogăcean, A. R. Biriş, M. Coroş, M. C. Roşu, L. Mageruşan, G. Katona and S. Pruneanu, Talanta, 2016, 148, 511–517.
15. Q. Huang and W. J. Weber, Environ. Sci. Technol., 2005, 39, 6029–6036.
16. R. Pang, M. Li and C. Zhang, Talanta, 2015, 131, 38–45.
17. Y. B. Feng, L. Hong, A. L. Liu, W. D. Chen, G. W. Li, W. Chen and X. H. Xia, Int. J. Environ. Sci. Technol., 2015, 12, 653–660.
18. W. Tu, J. Lei, G. Jian, Z. Hu and H. Ju, Chem.–Eur. J., 2010, 16, 4120–4126.
19. G. Wang, X. Shen, B. Wang, J. Yao and J. Park, Carbon, 2009, 47, 1359–1364.
20. V. D. Pham, J. Lagoute, O. Mouhoub, F. Joucken, V. Repain, C. Chacon, A. Bellec, Y. Girard and S. Rousset, ACS Nano, 2014, 8, 9403–9409.
21. M. S. Dresselhaus, A. Jorio, M. Hofmann, G. Dresselhaus and R. Saito, Nano Lett., 2010, 10, 751–758.
22. K. S. Subrahmanyam, S. R. C. Vivekchand, A. Govindaraj and C. N. R. Rao, J. Mater. Chem., 2008, 18, 1517–1523.
23. W. Tu, J. Lei, S. Zhang and H. Ju, Chem.–Eur. J., 2010, 16, 10771–10777.
24. M. Coros, F. Pogacean, M. C. Rosu, C. Socaci, G. Borodi, L. Magerusan, A. R. Biris and S. Pruneanu, RSC Adv., 2016, 6, 2651–2661.
25. S. Maldonado, S. Morin and K. J. Stevenson, Carbon, 2006, 44, 1429–1437.
26. G. P. Lewis, J. Clin. Pathol., 1965, 18, 235–239.
27. J. Silver and B. Lukas, Inorg. Chim. Acta, 1983, 78, 219–224.
28. L. Z. Gao, J. Zhuang, L. Nie, J. B. Zhang, Y. Zhang, N. Gu, T. H. Wang, J. Feng, D. L. Yang, S. Perrett and X. Y. Yan, Nat. Nanotechnol., 2007, 2, 577–583.
29. S. Li, H. Li, H. Zhang, Z. Yang and B. Wang, Dyes Pigm., 2016, 125, 64–71.
30. S. A. Snyder, P. Westerhoff, Y. Yoon and D. L. Sedlak, Environ. Eng. Sci., 2003, 20, 449–469.
31. N. Caza, J. K. Bewtra, N. Biswas and K. E. Taylor, Water Res., 1999, 33, 3012–3018.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15414j

---