Cubic boron nitride with an intrinsic peroxidase-like activity

Abstract

Inorganic artificial enzymes have been developed as potential candidates to naturally occurring enzymes, and three inorganic enzyme mimics, noble metals, metal oxides and carbon materials, have been reported so far. Here, we reported the inorganic enzyme mimic of nitride-based materials. We demonstrated that cubic boron nitride (c-BN) as an enzyme mimic showed intrinsic peroxidase-like activity towards classical colorimetric substrates in the presence of hydrogen peroxide (H₂O₂). The Michaelis–Menten kinetics studies indicated that the catalytic efficiency of c-BN is superior to its natural peroxidase counterparts. We also established that the peroxidase-like activity of c-BN is induced by catalyzing the decomposition of H₂O₂ and generating hydroxyl radicals (˙OH). Based on the color reaction, a strategy was developed for H₂O₂ and glucose quantitative detection with high sensitivity. A reactor was constructed by entrapping c-BN in a porous platform and the peroxidase mimic immobilization for removal of organic pollutants was successfully conducted. Additionally, c-BN can be re-used up to 5 times and retain its catalytic activity after incubation at extremes of pH and temperature. These findings open the door for the application of c-BN as a catalyst and the development of nitride-based materials in the enzyme-mimics field.

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1. Introduction

Enzymes, biocatalysts that participate in almost all biological processes in organisms, demonstrate outstanding catalytic capability for specific substrates and reactions.^1,2 However, their practical applications are severely restricted because of some disadvantages, such as a requirement for mild reaction conditions, low operational stability and high costs in the preparation and purification.^3,4 Recently, nanomaterial-based artificial enzymes have been developed as potential candidates to naturally occurring enzymes. For instance, Yan et al. reported that Fe₃O₄ nanoparticles show an intrinsic peroxidase-like activity.⁵ Afterward, some other nanomaterials, such as cerium oxide nanoparticles,⁶ vanadium oxide nanowires,⁷ gold nanoparticles,⁸ Au@Pt nanorods,⁹ carbon nanodots and graphene oxide were also reported to possess enzyme-like activity.^10,11 On basis of this specific characteristic, these enzyme mimics exhibit promising applications in a variety of fields including biosensing, immunoassays, cancer diagnostics and wastewater treatment.⁴ Although some advances have been made in mimetic enzymes, new inorganic materials with enhanced catalytic activity, high stability and efficient recycling have still received great interests to replace conventional enzyme systems for practical applications.¹² According to the chemical constituent characteristics, researchers have reported three inorganic enzyme mimics so far: noble metals, metal oxides and carbon materials.⁴

Cubic boron nitride (c-BN), known as one of the hardest materials, has been widely applied in the field of mechanical engineering.¹³ Furthermore, c-BN is a wide-gap semiconductor and can be doped to form p–n junctions, serving as light-emitting diodes,¹⁴ and c-BN has one of the highest thermal conductivities making it a promising heat sink for semiconductor lasers and microwave devices.¹⁵ Since c-BN exhibits remarkable stability, showing reactivity only at high temperatures and pressures, it has been regarded as a biologically and chemically inert material.^16,17 However, no studies have reported about the catalytic activity of c-BN.

Here, for the first time, we demonstrate that c-BN possesses the intrinsic peroxidase-like activity. In this case, we find out that c-BN can catalyze the oxidation of peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H₂O₂ to produce a blue color reaction. The Michaelis–Menten kinetics shows that c-BN has even higher efficiency for H₂O₂ and TMB than that of the natural enzyme, horseradish peroxidase (HRP), respectively. We also establish that the peroxidase-like activity of c-BN is induced by its ability to catalyze the decomposition of H₂O₂ and generating hydroxyl radicals (˙OH). Hexagonal boron nitride (h-BN) is another allotrope of boron nitride.^18–20 However, comparative experiments show that no color reactions of h-BN are observed in the presence of TMB and H₂O₂, which indicates that the peroxidase-like activity is mainly determined by c-BN crystal structure. On the base of the color reaction, a strategy was developed for H₂O₂ and glucose detection with high linearity and sensitivity. In addition, c-BN can be re-used up to 5 times and retains its catalytic activity after incubation in extremes of pH and temperature. On account of its large particle size and slight solubility in water, c-BN is explored to mimic the functions of immobilized peroxidase. By enclosing c-BN in a microporous polyethersulfone (PES) membrane, an immobilized enzyme-mimic reactor is constructed to remove organic substrates from wastewater. Therefore, these studies open the door for the application of c-BN as a catalyst and the development of nitride-based materials in the enzyme-mimics field.

2. Experimental

2.1. Materials preparation

Cubic boron nitride (c-BN, <1 μm) was obtained from Element Six Company. Cubic boron nitride (c-BN, 4–8 μm), hexagonal boron nitride (5–20 nm) and rhodamine B were purchased from Alfa Aesar. Hexagonal boron nitride (50 nm, 99.9%) was purchased from Shanghai Chaowei nanotechnology Co. Ltd. Hexagonal boron nitride (h-BN, ∼1 μm, 98.5%), 3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), o-phenylenediamine (OPD), glucose oxidase (100 U mg⁻¹) and NaOH (≥98.0%) were purchased from Aladdin Industrial Corporation. Hydrogen peroxide (30%), Na₂HPO₄ (≥99.0%), Citric acid (≥99.5%), Na₂CO₃ (≥99.8%), NaHCO₃ (≥99.5%), hydrochloric acid (36%) and glucose were obtained from Guangzhou Chemical Reagent Factory. Terephthalic Acid (>99.0%) and 2-hydroxyterephthalic acid (>98.0%) were purchased from Tokyo Chemical Industry Co. Ltd. Millipore water (18.2 MΩ) was used throughout the experiments.

2.2. Characterizations

XRD was performed using a Rigaku D/Max-IIIA X-ray diffractometer with Cu Kα radiation (λ = 1.54056 Å, 40 kV, 20 mA) at a scanning rate of 2° s⁻¹. SEM images and SEM-based energy-dispersive X-ray (EDX) measurements were taken on a Quanta 400F field-emission scanning electron microscope operated at 10 kV. FTIR spectra was recorded using a Bruker EQUINOX55 spectrometer coupled with an IR microscope. The surface analysis was performed by X-ray photoelectron spectroscopy (XPS) using a Thermo-VG Scientific ESCALAB 250 spectrometer. Photoelectrons were generated by mono Al Kα X-ray radiation of energy 1486.6 eV and the calibration was based on the C 1s with the binding energy at 284.8 eV. Fluorescence spectroscopy was performed on Hitachi F-4500 Fluorescence spectrophotometer. UV-vis spectra of the samples were obtained using a UV 3150 spectrophotometer (Shimadzu, Japan). HPLC analysis was carried out in an Agilent 1200 series HPLC (Agilent, USA) using a fluorescence detector (E_x = 315 nm, E_m = 425 nm). The analytical column used was an Eclise Plus C18 (4.6 × 250 mm, 5 μm). The mobile phase was a 100 mmol L⁻¹ potassium hydrogen phosphate containing 2% of KCl (pH 4.37). The injection volume was 10 μL.

2.3. Modification of c-BN

c-BN was modified by ion implantation and thermal annealing, respectively. In an ion implantation experiment, the sample was treated by ions at energy of 200 keV and doses of 1 × 10¹⁵ cm⁻², using an ion implanter. The implanted ion species are helium, boron and nitrogen, respectively. In an annealing experiment, c-BN was subjected to heat treatment at 800 °C for 2 hours. The annealing is done in a nitrogen atmosphere and air, respectively.

2.4. Kinetic analysis

Steady-state kinetic assays were carried by measuring the absorbance change of the c-BN/TMB/H₂O₂ system at 652 nm (ε = 39 000 M⁻¹ cm⁻¹) for 5 minutes, using a UV 3150 spectrophotometer (Shimadzu, Japan). Experiments were performed at 45 °C, using 0.25 mg ml⁻¹ c-BN in a reaction volume of 4 ml citrate–phosphate buffer solution (100 mM citric acid, 200 mM dibasic sodium phosphate, pH 4.0) with 1 mM TMB as substrate, and H₂O₂ was 100 mM, unless otherwise stated. H₂O₂ was added to start the reaction while c-BN was removed from the sample by a syringe filter, prior to recording the absorbance. The apparent kinetic parameters were calculated based on the Michaelis equation υ = V_max × [S]/(K_m + [S]), where υ is the initial velocity, V_max is the maximal reaction velocity, [S] is the concentration of the substrate, and K_m is the Michaelis–Menten constant. To confirm the reproducibility, kinetic assays were performed triplicate.

2.5. Detection of hydroxyl radicals by fluorimetry

Terephthalic acid (TA) was used as a fluorescence probe for tracking of ˙OH. In the experiment, a standard solution of sodium salt of TA (Na₂TA) with the concentration of 25 mM was prepared by dissolving 830.7 mg of TA in 200 ml of NaOH (62.5 mM). 0.4 ml of Na₂TA solution was added into a mixture of 3.6 ml citrate–phosphate buffer solution (pH 6.0) with 0.25 mg ml⁻¹ c-BN and 100 mM H₂O₂. Followed by 3.5 hours incubation in the dark at 37 °C, the resulting solution was filtered for fluorescence measurement. The excitation and emission wavelengths were 315 and 425 nm, respectively.

2.6. Re-utilization assays

The re-utilization of the catalyst was tested using a scale-up of the reaction, in which 1 mg of c-BN were used for the final concentration of 0.25 mg ml⁻¹. The reactions were carried out in citrate–phosphate buffer solution (100 mM citric acid, 200 mM dibasic sodium phosphate, pH 4.0), using 1 mM of TMB, and H₂O₂ (10 mM) was added to start the reaction. The absorbance at 652 nm was measured using a UV 3150 spectrophotometer (Shimadzu, Japan) after which the catalyst was spun by centrifugation (10 000 rpm for 10 min). c-BN was washed with MilliQ water, and a new reaction cycle (a total of 5) was performed following the same procedure.

2.7. Rhodamine B removal experiment

Experiments were performed using 1 mg ml⁻¹ c-BN in a reaction volume of 4 ml citrate–phosphate buffer solution (pH 2.5) with 245 mM H₂O₂ and 25 μM RhB. Followed by 5 hours incubation in the dark at room temperature, the resulting solution was filtered for UV-vis absorbance characterization. The peak absorbance of rhodamine B lies at 570 nm.

2.8. Design and operation of immobilized enzyme-mimic reactor

An immobilized enzyme-mimic reactor consists of three syringe filters and a cylindrical tube (the barrel of a syringe). These filters were (PES membrane, pore size 0.2 μm, diam. 25 mm) filled with 10 mg c-BN respectively and assembled end to end in sequence. A mixed solution of 0.4 ml RhB (1 mM), 4.6 ml H₂O₂ (30%) and 10 ml citrate–phosphate buffer solution (pH 4.0) was pouring into the tube and flowed through the filters slowly due to the forces of gravity. During this process, the solution was catalyzed by the immobilized c-BN. The resulting solution could be obtained from the orifice of filters. Additionally, the flow rate of the solution could be increased by plunger expelling or decreased by the tap controls.

3. Results and discussion

3.1. Characterization of c-BN

Clearly, the XRD pattern of the sample in Fig. 1a demonstrates the presence of c-BN (JCPDS no. 79-0623). The corresponding scanning electron microscopy (SEM) analysis in Fig. 1b shows that c-BN particles are of irregular-particle shape. To obtain a reliable size distribution, we carry out a statistical analysis of 250 particles according to SEM data and find out that the diameter of c-BN particles is mainly distributed in 100–800 nm with the average size of approximately 420 nm (Fig. 1c). Complementary analytical data on c-BN particles using energy-dispersive analysis of X-rays (EDX) confirm that only boron and nitrogen are present (Fig. 1d).

Fig. 1 (a) XRD pattern of c-BN. All reflections can be indexed to cubic form boron nitride. (b) SEM image of c-BN. (c) Size distribution analysis from 250 random particles. (d) Corresponding EDX spectra showing the presence of boron (B) and nitrogen (N).

3.2. Peroxidase-like activity

c-BN particles are capable of catalyzing typical peroxidase reactions, as shown in Fig. 2a, using 3 typical peroxidase substrates, including 3,3′-diaminobenzidine (DAB), o-phenylenediamine (OPD) and 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H₂O₂ to produce a brown (maximum absorbance at 465 nm), orange (maximum absorbance at 450 nm) and blue (maximum absorbance at 652 nm) color reaction, respectively. The reactions are also carried out in the absence of c-BN, TMB or H₂O₂ respectively. No color change is observed (Fig. S1†), suggesting that all these components are necessary for the reaction to occur. For a comparison, the oxidation activity towards TMB of another kind of commercially available c-BN (4–8 μm) is also tested (Fig. S1†). Its oxidation activity is also visualized in conditions used in the above experiments. Thus, these results confirm that c-BN particles possess the intrinsic peroxidase-like activity. Fig. 2b illustrates the oxidation of TMB catalyzed by c-BN in the presence of H₂O₂.

Fig. 2 c-BN shows the peroxidase-like activity. (a) The c-BN catalyzes, in the presence of H₂O₂, the oxidation of various peroxidase substrates: (1) DAB, (2) OPD and (3) TMB. (b) Schematic illustration of peroxidase activities of c-BN. (c and d) The peroxidase-like activity of c-BN is pH and temperature dependent. Experiments were carried out using 1 mg c-BN in a reaction volume of 4 ml, in citrate–phosphate buffer, with 1 mM TMB as substrate. The H₂O₂ concentration was 100 mM and pH was 4.0, and the temperature was 45 °C, unless otherwise stated. The maximum point in each curve (c and d) was set as 100%. From (c), c-BN shows an optimal pH of 3.5–4.5. From (b), c-BN shows an optimal temperature around 40–50 °C.

The peroxidase-like activity of c-BN is, as for HRP, dependent on pH and temperature (Fig. 2c and d). It is measured while varying pH from 2 to 10, temperature from 25 °C to 60 °C, indicating the optimal pH and temperature are approximately pH 4.0 and 45 °C respectively, adopted as standard conditions for subsequent analysis of the c-BN activity.

3.3. Apparent steady-state kinetics

The apparent steady-state kinetics is investigated for the reaction catalyzed by c-BN. Similar to the enzyme-catalyzed reaction, the typical Michaelis–Menten curves are observed for c-BN (Fig. 3a and b). Kinetic parameters are obtained by fitting to the Michaelis–Menten model. As shown in Table 1, the apparent K_m value of c-BN with TMB as the substrate is much lower than that for HRP, suggesting that c-BN has a higher affinity for TMB than HRP. On the other hand, the apparent K_m value of c-BN for H₂O₂ is higher than HRP, indicating that higher H₂O₂ concentration is required to reach maximal activity for c-BN. Furthermore, the catalytic constant K_cat reveals that c-BN shows a level of catalytic efficiency 10 times higher for H₂O₂ and TMB than HRP, respectively.⁵

Fig. 3 Apparent steady-state kinetic study of c-BN. (a–d) The velocity (v) of the reaction was measured using 1 mg c-BN (a and b) in 4 ml of phosphate pH 4.0 at 45 °C. (a) The concentration of H₂O₂ was 100 mM and the TMB concentration was varied. (b) The concentration of TMB was 1 mM and the H₂O₂ concentration was varied. The error bars represent the standard deviation of three measurements. (c and d) Double-reciprocal plots of activity of c-BN at a fixed concentration of one substrate versus varying concentration of the other substrate for H₂O₂ and TMB.

Table 1 Comparison of the kinetic parameters of c-BN and HRP. [E] is the concentration of HRP (or c-BN), K_m is the Michaelis constant, V_max is the maximal reaction velocity and K_cat is the catalytic constant, where K_cat = V_max/[E]

Catalyst	[E] (10^−12 M)	Substrate	Km (mM)	Vmax (10^−8 M s^−1)	Kcat (10^4 s^−1)	References
HRP	25	TMB	0.434	10.0	0.400	5
HRP	25	H2O2	3.70	8.71	0.348
c-BN	3.10	TMB	0.157 ± 0.043	18.54 ± 1.41	5.98 ± 0.45	This work
c-BN	3.10	H2O2	10.88 ± 1.96	10.69 ± 1.18	3.45 ± 0.38

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The peroxidase activity of c-BN over a range of TMB and H₂O₂ concentrations is measured. Fig. 3c and d show double reciprocal plots of initial velocity versus one substrate concentration, obtained for a range of concentrations of the other substrate. The parallel slope of the lines reveals a ping–pong mechanism, as is characteristic of HRP. This then indicates that, like HRP, c-BN bind and react with the first substrate, releasing the first product before reacting with the other substrate.⁵

3.4. Mechanism of the catalytic reaction

Generally, the catalytic pathways of peroxidase-like activity can be sorted into the electron transfer process or the generation of ˙OH.²¹ To clarify the catalytic mechanisms of c-BN, fluorescence experiments are carried out to detect ˙OH during the reaction. Terephthalic acid (TA) is used as a fluorescence probe for tracking of ˙OH because it could capture ˙OH and generate 2-hydroxyterephthalic acid (HTA), which emitting the unique fluorescence around 425 nm (Fig. 4a).¹⁰ Fig. 4b shows the fluorescence change in the mixed solution of c-BN, TA and H₂O₂. After 12 hours reaction, compared with control experiments, remarkable fluorescence enhancement at 425 nm indicates the presence of ˙OH.¹⁰

Fig. 4 (a) The reaction between ˙OH and TA. (b) Fluorescence spectra of phosphate solutions (pH 6.0) containing only TA, TA and c-BN, TA and H₂O₂, or TA, c-BN and H₂O₂ after a reaction time of 12 hours. The TA, H₂O₂, and c-BN concentrations were 2.5 mM, 979 mM, and 0.5 mg ml⁻¹, respectively. (c) Possible mechanism for ˙OH generation in the c-BN/H₂O₂ system.

To further confirm the fluorescent product is HTA, High-performance liquid chromatography (HPLC) with fluorescence detection method is employed.^22,23 Fig. S2† shows the HPLC chromatograms of a HTA standard, in which the fluorescent sample and its blanks with excitation at 315 nm and emission at 425 nm. The retention time (RT) of the fluorescent sample is 4.1 min (Fig. S2d†), identical to that of the HTA standard (Fig. S2e†), which suggests the fluorescent product is HTA and the ˙OH are produced in the catalytic reaction for certain.²³

The possible mechanism for ˙OH generation in the c-BN/H₂O₂ system is proposed (Fig. 4c). It is assumed that the H₂O₂ is firstly absorbed on the surface of c-BN. Subsequently, the O–O bond of H₂O₂ might be broken up into double ˙OH facilitated by c-BN.⁹ The generated ˙OH can be stabilized at the surface of c-BN, contributing to the oxidation of the peroxidase substrates.⁸

3.5. Origin of peroxidase-like activity

The surface properties of catalysts may play an important role on their catalyzed activity.⁹ In order to analyze the surface composition and chemical states of c-BN, X-ray photon electron spectroscopy (XPS) are conducted. The XPS spectrum of c-BN (Fig. S3a†) demonstrates that c-BN particles are mainly composed of B (48.54%) and N (37.11%) elements. The observation of O and C may be mainly result from the surface contamination of hydrocarbon impurities possessing C–C and C=O bonds.²⁴ The high resolution XPS spectrums also indicates the presence of BN_xO_y species and C–N bonds in limited amounts (Fig. S3b–e, for details, see ESI†).²⁵ However, the signals of these bonds and species do not show up in the Fourier transform infrared (FTIR) spectroscopy (Fig. S3f†). The FTIR spectroscopy only shows the existence of O–H (around 3400 cm⁻¹), derived from water on the surface, and B–N (around 1100 cm⁻¹), suggesting that the surface of c-BN is almost unmodified.²⁶

The XPS spectrum shows the ration of B to N is 1.31, revealing the existence of nitrogen vacancy defects on the surface.^27,28 To investigate the effect of surface defects on its peroxidase-like activity, in our case, ion implantation and thermal annealing are employed to tune the concentrations of surface defects of c-BN. The typical absorbance peak of oxidation products of TMB is at 652 nm (Fig. 5a). Clearly, the absorbance of the ion-implanted-c-BN/TMB/H₂O₂ system is higher, indicating that the activity of ion-implanted-c-BN is enhanced compared with raw materials. This is mainly due to the increase of defects concentrations on the surface of c-BN caused by ion implantation.²⁹ At the same level of implanted energy and dose, nitrogen ion with the larger particle size, in comparison to boron and helium, causes more defects. Hence the nitrogen-implanted-c-BN has the higher catalytic activity. On the other hand, since atoms migrate in the crystal and the number of defects decreases in annealing, the treated c-BN exhibits reduced activity (Fig. 5b).³⁰ Particularly, the air-treated c-BN shows lower activity than that of c-BN treated by nitrogen atmosphere, indicating that the oxide coating of the surface has negative effects on the peroxidase-like activity.

Fig. 5 (a) The UV-visible absorption spectra of TMB with H₂O₂ and c-BN treated by boron, nitrogen or helium ion implantation. (b) The UV-visible absorption spectra of TMB with H₂O₂ and c-BN treated by annealing in the air or nitrogen atmosphere at 800 °C for 2 hours.

h-BN is another allotrope of boron nitride and its reactivity is relatively similar to c-BN.^18–20 Therefore, it might also have the peroxidase-mimic activity. h-BN particles with different sizes (5–20 nm, 50 nm, 1 μm and 1–2 μm) are adopted in our experiment. However, no color reaction of h-BN is observed in the presence of TMB and H₂O₂ (Fig. S4†). XRD, SEM, EDX and FTIR spectrum provide compelling evidences (these h-BN samples are all hexagonal form boron nitride with high purity (Fig. S5†)).³¹ The XPS spectrum and the high resolution XPS spectrums of B 1s, N 1s, C 1s and O 1s of h-BN are relatively similar to that of c-BN.^24,25 In addition, the defects are also found on the surface of h-BN (Fig. S6†).²⁸ Therefore, these results reveal that the peroxidase-like activity is an intrinsic property of c-BN, which is probably originated from its special crystal structure rather than the surface contaminants or defects on its surface. Since the numbers of dangling bonds and the atom arrangement manners vary from plane to plane, the exposed planes of c-BN are different from that of h-BN, which may determine the distinct differences existed in the peroxidase-like activity between them.³² Further research about the effects of crystal structure on the peroxidase-like activity is needed in the future.

3.6. Applications of c-BN enzyme mimic

c-BN is an ultra-stable inorganic material, even in extreme environment.^16,17 To test this, c-BN particles are firstly incubated at a range of temperatures (4, 15, 37, 45, 60, 75, 90 °C) and a range of values of pH (0–12) for 2 hours, and then measured in the standard conditions (pH 4.0 and 45 °C). Interestingly, c-BN is indeed found to remain stable over a wide range of temperatures from 4 to 90 °C, and pH from 1 to 12 (Fig. S7a and b†). Furthermore, the re-utilization of c-BN is also test in a series of catalytic cycles. Firstly, the catalytic reaction is performed in the optimized conditions. Subsequently, c-BN is recovered, centrifuged, and fresh substrates are added. Fig. 7a shows that after 5 cycles, c-BN particles partially retain their catalytic activity with some decrease after each cycle. Between each catalytic step an average decay in activity of 6.7% is observed. Thus, this highlights the excellent catalytic properties of c-BN. In general, the robustness and easy recovery of c-BN make it suitable for a broad range of applications in the environmental chemistry and industrial manufacture fields.

Because the catalytic activity of c-BN is H₂O₂ concentration dependent, the system discussed above could be utilized to quantitatively detect H₂O₂. In the optimized conditions, the developed method is used for H₂O₂ detection. As shown in Fig. 6a, the absorbance of this system increases with increasing H₂O₂ concentration. The calibration graph of the absorbance at 652 nm to H₂O₂ concentration is linear in the range of 10 to 200 μM with a detection limit of 8 μM. By coupling the oxidation of glucose catalyzed by glucose oxidase (GOD), the colorimetric method is further developed for quantitative analysis of glucose. Fig. 6b displays the linear response of the absorbance versus glucose concentration in the range of 10 to 100 μM with a detection limit of 8 μM.

Fig. 6 (a) Linear calibration plot between the absorbance at 652 nm and concentration of H₂O₂. The insert shows the dependence of the absorbance at 652 nm on the concentration of H₂O₂ in the range 10 to 300 μM. (b) Linear calibration plot between the absorbance at 652 nm and concentration of glucose. The insert shows the dependence of the absorbance at 652 nm on the concentration of glucose in the range 10 to 300 μM.

Advanced oxidation process (AOP), an environmentally friendly chemical treatment procedure, is designed to degrade organic materials in waste water by oxidation through reactions with ˙OH.³³ Considering the c-BN/H₂O₂ system could efficiently produce ˙OH, it can be employed in an AOP to remove organic pollutants in the presence of H₂O₂. Rhodamine B (RhB), a widely used staining agent, is chosen as the model compound to assess the ability of c-BN for pollutants removal.³⁴ As shown in Fig. S8,† the absorbance of RhB at 570 nm changed obviously upon incubation with H₂O₂ and c-BN for 5 h. The removal efficiency of RhB is 40%, indicating that c-BN exhibits potential applications in AOP.

The immobilized enzyme is known as an enzyme that is attached to an inert, insoluble material such as semipermeable polymer membrane. The immobilization allows enzymes to be held in place throughout the reaction, following which they are easily separated from the products and may be used again.³⁵ Since c-BN has large particle size and slight solubility in water, it can be explored to mimic the functions of immobilized peroxidase. By enclosing it in a microporous PES membrane, a reactor model is developed (Fig. 7b). We coin the terms “immobilized enzyme-mimic reactor” to describe this equipment. It is capable of carrying out the removal of organic substrates such as RhB from wastewater. In this reactor, the waste water suffered a more sufficient contact to the catalyst compared to free c-BN. Furthermore, no separation techniques are needed to reuse the catalyst after the reaction, making it a far more efficient process. Within 60 minutes, 4 ml of the treated RhB solution is produced, with a removal efficiency of 86%. This immobilized enzyme-mimic reactor shows potential in practical application for catalyzed pollutants removal.

Fig. 7 (a) Catalytic activity of c-BN over cycles of re-utilization using identical reaction conditions and a constant oxidation rate. (b) Image of the immobilized enzyme-like reactor. The insert shows removal of RhB treated by the immobilized enzyme-like reactor.

4. Conclusion

In summary, we have for the first time demonstrated that c-BN possesses the intrinsic peroxidase-like activity. In the presence of H₂O₂, it can catalyze the oxidation of typical peroxidase substrates such as DAB, OPD and TMB to produce the same color reaction as HRP. Its catalysis is strongly dependent on pH, temperature and H₂O₂ concentration. The Michaelis–Menten kinetics indicated that c-BN has higher catalytic activity for TMB and a level of catalytic efficiency 10 times higher for H₂O₂. Like HRP, the reaction follows a ping–pong catalytic mechanism. The catalytic pathway of activity was confirmed as its ability of production of ˙OH upon reaction with H₂O₂. On the base of the color reaction, c-BN has been applied in H₂O₂ and glucose detection with high linearity and sensitivity. In addition, c-BN can be re-used up to 5 times and retains its catalytic activity after incubation in extremes of pH and temperature. Finally, c-BN was explored to mimic the functions of immobilized peroxidase. By enclosing it in a microporous membrane, an immobilized enzyme-mimic reactor was constructed to remove RhB from water successfully. Therefore, these studies suggested that c-BN could be expected to be a potential candidate to naturally occurring enzymes.

Acknowledgements

The National Basic Research Program of China (2014CB931700) and State Key Laboratory of Optoelectronic Materials and Technologies supported this work.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14856e

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