Fe₃O₄ nanoparticles entrapped in the inner surfaces of N-doped carbon microtubes with enhanced biomimetic activity

Abstract

Tubular structured composites have attracted great interest in catalysis research owing to their void-confinement effects. In this work, we synthesized a pair of hollow N-doped carbon microtubes (NCMTs) with Fe₃O₄ nanoparticles (NPs) encapsulated inside NCMTs (Fe₃O₄@NCMTs) and supported outside NCMTs (NCMTs@Fe₃O₄) while keeping other structural features the same. The impact of structural effects on the catalytic activities was investigated by comparing a pair of hollow-structured nanocomposites. It was found that the Fe₃O₄@NCMTs possessed a higher peroxidase-like activity when compared with NCMTs@Fe₃O₄, demonstrating structural superiority of Fe₃O₄@NCMTs. Based on the excellent peroxidase-like catalytic activity and stability of Fe₃O₄@NCMTs, an ultra-sensitive colorimetric method was developed for the detection of H₂O₂ and GSH with detection limits of 0.15 μM and 0.49 μM, respectively, which has potential application value in biological sciences and biotechnology.

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Introduction

In recent years, nanozymes, as a type of nanomaterials with enzyme-like catalytic activity, have attracted much attention owing to their advantages of low preparation cost, high stability, and adjustable catalytic activity.^1–4 As is well known, Fe₃O₄ magnetic nanoparticles (MNPs) are typical artificial mimics with peroxidase catalytic activity.⁵ They have excellent biocompatibility and strong magnetism, showing broad application prospects in biocatalysis, sensing, drug delivery, medical imaging, etc.^6–12 Despite these achievements, these Fe₃O₄-based nanozymes have been used in acid buffer solutions. As is well known, Fe₃O₄ nanoparticles (NPs) are unstable and easily etched in acid solutions, greatly hindering their further application in the sensing field. Therefore, it is highly desired to find a suitable support to protect Fe₃O₄ NPs while retaining high catalytic activity.

Recently, hollow structured nanocomposites containing a void space inside a distinct shell have attracted much attention in the fields of catalysis owing to their unique structures such as high specific surface area, available pore space, adjustable shell pore structure, and adjustable pore space.^13–15 From the perspective of chemical and biomolecular engineering, nanomaterials that encapsulate active species inside hollow structures can be considered as nanoreactors and used in a series of heterogeneous catalytic processes.¹⁶ The shell of these nanoreactors exhibits a rich porous structure¹⁷ and high specific surface area,¹⁸ which protects the internally loaded active species from sintering, leaching, and aggregation. In addition, the chemical reactions in a given cavity show excellent catalytic activity and high selectivity due to the limitation of nanoreactors.¹⁹ For instance, Yang²⁰et al. reported a facile route to prepare magnetic tubular nanoreactors composed of a mesoporous SiO₂ shell with iron-noble metal nanoparticles inside, which could prevent the leaching of Pd NPs and thus improve the catalytic activity and stability of the catalyst. Peng²¹et al. reported the synthesis of TiO₂-encapsulated Au (Au@TiO₂) yolk–shell nanostructures with enhanced catalytic activity for the detection of glucose. Dong²²et al. also found that PdCu NPs encapsulated inside hollow carbon spheres (HCSs) showed better catalytic performance and stability in liquid phase hydrogenation reactions than those supported outside HCS. Thus, the encapsulation of active species in hollow nanomaterials provides an ideal model for the rational design and development of efficient catalysts employed in the biocatalytic field.

On the other hand, nitrogen-doped carbon materials have aroused great interest due to their excellent electronic properties, electrical conductivity and high catalytic activity.^23–25 Nitrogen doping not only endows carbon materials with better electrical conductivity but also produces many anchoring sites,²⁶ thus forming multifunctional composites. On the basis of the findings, we synthesized a pair of hollow N-doped carbon microtubes (NCMTs) with Fe₃O₄ NPs encapsulated inside of NCMTs (Fe₃O₄@NCMTs) and supported outside of NCMTs (NCMTs@Fe₃O₄), respectively, while keeping other structural features the same. It was found that the Fe₃O₄@NCMTs possessed a higher peroxidase-like activity when compared with NCMTs@Fe₃O₄, demonstrating the structural superiority of Fe₃O₄@NCMTs. In addition, a label-free, highly sensitive, and selective colorimetric approach for the detection of H₂O₂ and GSH was established, suggesting promising potential diagnostics in real-life applications.

Experimental

Material

Pyrrole was obtained from the Alfa Aesar Chemical Company. Ammonium molybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O), concentrated nitric acid, ammonium persulfate ((NH₄)₂S₂O₈), ammonia solution (28–30%), ammonium ferric sulfate dodecahydrate (NH₄Fe(SO₄)₂·12H₂O) 3,3′,5,5′-tetramethylbenzidine (TMB) and 30% H₂O₂ were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Other chemical reagents were purchased from the Shanghai chemical reagent company. All reagents were used without further purification.

Synthesis of MoO₃

The MoO₃ microrods were synthesized according to our previous report. Typically, (NH₄)₆Mo₇O₂₄·4H₂O (1 g) was dissolved in a 25 mL mixed solution of 65% HNO₃ and deionized H₂O with a volume ratio of 1 : 5. Then, the solution was transferred into a Teflon-lined stainless-steel autoclave (50 mL capacity) and heated at 180 °C for 20 h. After cooling, the products were collected and washed with water and ethanol several times and then dried at 60 °C overnight.

Synthesis of MoO₃@FeOOH

The preparation of MoO₃@FeOOH was based on a modified method. Firstly, the as-synthesized MoO₃ microrods (0.288 g) were dispersed into a 100 mL mixture of ethanol and water with a volume ratio of 1 : 9. Then, 80 mL of NH₄Fe(SO₄)₂·12H₂O (1.928 g) aqueous solution was added into the above suspension and the mixture solution continued stirring at 70 °C for 3 h. Finally, the orange solution was filtrated and then dried at 60 °C.

Synthesis of MoO₃@FeOOH@PPy

The MoO₃@FeOOH@PPy composites were prepared through an in situ oxidative polymerization. Freshly prepared MoO₃@FeOOH (0.1 g) microrods were homogenously dispersed into a mixed solution of 40 mL deionized water and 5 mL anhydrous ethanol. With vigorous stirring, 0.1 mL of pyrrole was added to the white suspension and the stirring continued for 0.5 h. Then, 0.329 g of (NH₄)₂S₂O₈ (dissolved in 5 mL of deionized water) was dropped into the above mixture and the polymerization was performed for 4 h. Finally, the black solution was filtrated and then dried at 60 °C.

Synthesis of Fe₃O₄@NCMTs

The as-synthesized MoO₃@FeOOH@PPy (50 mg) and ammonia aqueous solution (1 mL) were dispersed in 20 mL of deionized water. After stirring for 10 min, the products were collected and washed with water and ethanol several times and then dried at 60 °C overnight. Subsequently, the obtained tubular FeOOH@PPy composites were annealed at different temperatures (500, 700 and 900 °C) under N₂ gas for 5 h to obtain the Fe₃O₄@NCMTs composites, which were denoted as Fe₃O₄@NCMTs-500, Fe₃O₄@NCMTs-700 and Fe₃O₄@NCMTs-900. As a comparison, NCMTs@Fe₃O₄–700 was prepared as control samples, and the detailed experimental procedures are provided in the ESI.†

Peroxidase-like catalytic activities of Fe₃O₄@NCMTs-700

The catalytic activities of Fe₃O₄@NCMTs were performed using the following procedures. Typically, 10 μL of TMB solution (4 mM in anhydrous ethanol) and 10 μL of H₂O₂ (0.05 M) were added into 370 μL of acetate buffer solution (pH 4.0). Subsequently, Fe₃O₄@NCMTs dispersions calcined at different temperatures (10 μL, 1.0 mg ml⁻¹) were added to the above solution, respectively. After the mixture was incubated at 35 °C for 10 min, the absorbance at 652 nm was detected by UV-vis spectrophotometry.

Detection of H₂O₂ and glutathione using Fe₃O₄@NCMTs-700

For the detection of H₂O₂, 370 μL of acetate buffer solution (pH 4.0) adding varied concentrations of H₂O₂ (0–2500 μM) were mixed with 10 μL of Fe₃O₄@NCMTs-700 (10 μg mL⁻¹) and 10 μL of TMB (0.1 mM). After that, the absorbance changes at 652 nm were employed to generate a dose of H₂O₂ concentration-dependent responsive curve. The colorimetric detection of glutathione (GSH) is similar to that of H₂O₂. Typically, 10 μL of GSH with varied concentrations were added into the acetate buffer solution (360 μL, pH 4.0) containing Fe₃O₄@NCMTs-700 (10 μL, 10 μg mL⁻¹), TMB (10 μL, 0.1 mM in anhydrous ethanol) and H₂O₂ (10 μL, 1.25 mM). Then, the absorbance changes at 652 nm were monitored by UV-vis absorption spectroscopy. Furthermore, in order to evaluate the selectivity of the proposed colorimetric detection method, 100 mM of histidine (His), glucose (Glu), uric acid (UA), aspartic acid (Asp), and other ions (such as Mg²⁺, Ca²⁺, Na⁺, K⁺, and Cl⁻) were added into the above reaction system instead of GSH.

Characterization

The morphology and micro-structures of the samples were characterized by scanning electron microscopy (SEM, JEOL-4800) and transmission electron microscopy (TEM, JEOL-1011). The XRD pattern of each sample was recorded with a Shimadzu (Japan) D/Max-2500 diffractometer using a monochromatized X-ray beam with nickel-filtered Cu = Kα radiation. The nitrogen adsorption–desorption isotherms were measured on a Quanta chrome Auto sorb AS-1 instrument. The magnetic properties were collected using vibrating sample magnetometry (VSM, Micro Sense EV7) at room temperature. XPS of the Fe₃O₄@C state was measured using a Physical Electronics PHI 5600 XPS spectrophotometer with a monochromatic ALK (1486.6 eV) excitation source. The data on UV-vis adsorption was obtained by using a UV-2450 spectrophotometer (Shimadzu, Japan).

Results and discussion

Characterization of the as-prepared products

The general procedures for the fabrication of Fe₃O₄@NCMTs composite are schematically illustrated in Fig. 1(A). First, the MoO₃ microrods were prepared using a simple hydrothermal method. Subsequently, the FeOOH nanorods were uniformly grown on the surface of MoO₃ microrods via the hydrolysis of Fe³⁺. Next, the MoO₃@FeOOH microrods were coated with a uniform PPy layer by polymerization. Then, the MoO₃ core was etched in ammonia solution to obtain hollow FeOOH@PPy microtubes. Finally, followed by calcination under the N₂ atmosphere, the PPy layers were transformed into nitrogen-doped carbon microtubes. Simultaneously, the inner FeOOH was reduced to Fe₃O₄ by carbon sources from the carbonized PPy shell, resulting in the formation of 1D magnetic nitrogen-doped carbon microtubes (Fe₃O₄@NCMTs).

Fig. 1 (A) Schematic diagram of the synthesis process of Fe₃O₄@NCMTs. TEM images of (B) MoO₃, (C) MoO₃@FeOOH, (D) MoO₃@FeOOH@PPy, (E) FeOOH@PPy, and (F) Fe₃O₄@NCMTs-700. (G and H) HRTEM image and (I) elemental mapping images of Fe₃O₄@NCMTs-700.

SEM, TEM and XRD techniques were extensively applied to characterize different products. The as-prepared MoO₃ comprises highly uniform 1D nanorods with smooth surfaces and a diameter of 200–300 nm, as revealed in the SEM (Fig. S1A and B†) and TEM images (Fig. 1B). Fig. S2A† also shows all the diffraction peaks at 2θ values of 12.7°, 23.3°, 25.7°, 27.3°, 38.9°, 58.8°, and 67.5°, corresponding to the (0 2 0), (1 1 0), (0 4 0), (0 2 1), (0 6 0), (0 8 1), and (0 10 0) lattice planes of orthorhombic α-MoO₃ (JCPDS card no. 05-0508).²⁷ Uniformly rough and fluffy structures were observed for MoO₃@FeOOH, indicating the successful and full coating of FeOOH nanotubes on the MoO₃ surface (Fig. 1C and S1(C, D)†). The XRD pattern of MoO₃@FeOOH (Fig. S2B(a)†) is similar to that of MoO₃ and the diffraction peaks of MoO₃ are reduced after coating FeOOH, indicating that FeOOH microrods exist in an amorphous form and do not change the crystal characteristics of MoO₃. It was seen in Fig. 1D and S1(E, F)† that the rod-like structure of MoO₃@FeOOH also maintains its shape well during the coating of the PPy shell. Moreover, Fig. S2B(b)† displays that there is no obvious change in the XRD spectra of the material before and after coating PPy, which further confirms that the polymer coating plays a skeletal supporting role in the preparation of one-dimensional tubular composites. Afterwards, hollow tubular structures could be clearly observed in Fig. 1E and S1(G, H)†, indicating the formation of FeOOH@PPy intermediates by etching MoO₃ cores in ammonia solution, which were further converted into Fe₃O₄@NCMTs-700 composites by calcinations at 700 °C in nitrogen atmosphere. It was found in Fig. 1F that Fe₃O₄ NPs with an average size of 20 nm are uniformly doped in N-doped carbon microtubes. Furthermore, in the XRD patterns of FeOOH@PPy and Fe₃O₄@NCMTs-700 (Fig. S2B(c and d)†), only broad, amorphous peaks can be observed, and there are no other obvious characteristic peaks. This might be due to the relatively small particle size of the generated Fe₃O₄ NPs and the uniform distribution inside the tube. Fig. 1(G and H) shows the high-resolution transmission TEM of Fe₃O₄@NCMTs-700. Besides, the high-resolution TEM (HRTEM) techniques were employed to further clarify the microstructured composition of the as-prepared Fe₃O₄@NCMTs-700. The HRTEM images demonstrate that the spacings of the lattice stripes are 0.25 nm and 0.48 nm, corresponding to the (311) and (111) planes of Fe₃O₄,²⁸ respectively. In addition, it can be observed in the elemental mapping images (Fig. 1I) that Fe and O elements are evenly distributed along the inner side of the N-doped carbon hollow tube, further confirming the successful preparation of Fe₃O₄@NCMTs-700.

X-ray photoelectron spectroscopy was carried out to further analyze the surface electronic states and the chemical elemental composition of Fe₃O₄@NCMTs-700. As depicted in Fig. 2A, all the detected signal peaks of Fe, C, N and O elements are clearly observed in the survey XPS spectrum. For the Fe 2p spectrum (Fig. 2B), two peaks located at about 710.8 eV and 724.5 eV are indexed to Fe₃O₄, corresponding to Fe 2p_3/2 and Fe 2p_1/2, demonstrating the presence of Fe₃O₄ in the Fe₃O₄@NCMTs-700 composites.²⁹Fig. 2C shows the N 1s spectrum and the two peaks around 398.5 eV and 400.2 eV are consistent with pyridinic (C=N) and pyrrolic (C–N) nitrogen atoms, respectively. Also, the peak in the C 1s spectrum is identified as graphitic sp2-bonded amorphous carbon at 284.6 eV (ref. 30) (Fig. 2D), which indicates that the carbon layer obtained has a good graphitized structure after carbonization treatment.

Fig. 2 The XPS spectrum of Fe₃O₄@ NCMTs-700: (A) survey spectra, (B) Fe 2p, (C) N 1s, (D) C 1s. (E) The nitrogen adsorption/desorption isotherm of Fe₃O₄@ NCMTs-700 (the inset represents the corresponding pore-size distributions). (F) magnetization curves of Fe₃O₄@ NCMTs-700.

In addition, the specific surface area and porosity of Fe₃O₄@NCMTs-700 were further characterized by N₂ adsorption–desorption assays. Fig. 2E illustrates a typical type-IV curve indicating the presence of mesoporous properties in the as-prepared Fe₃O₄@NCMTs-700. The average pore size of the Fe₃O₄@ NCMTs-700 calculated from the adsorption branch centers is 14.21 nm and the total pore volume is 0.298 cm³ g⁻¹. Furthermore, the Fe₃O₄@NCMTs-700 composites demonstrated a high BET surface area of 141.59 m² g⁻¹, which might be attributed to the notched structure of 1D hollow NCMTs and the compact distribution of Fe₃O₄ NPs. Besides, the porous structures of Fe₃O₄@NCMTs-700 increased the amount of catalytic active sites on the surface and greatly enhanced the catalytic performance. Thus, the high surface area and porous structure are greatly beneficial for the subsequent catalytic application research. Additionally, to investigate the magnetic properties of the materials, the magnetic hysteresis loop properties were also measured by using a vibrating specimen magnetometer (VSM) at room temperature within a magnetic field of 6000 Oe. Fig. 2F represents the hysteresis loop of Fe₃O₄@NCMTs-700, and the magnetic saturation value is 14.47 emu g⁻¹, displaying good magnetic behavior and could be recycled and recovered with a magnet.

Different carbonization temperatures would cause uncertain amounts of volatiles to be released from the polymer, which ultimately affects the properties of composites.^31,32 Therefore, the influence of different carbonization temperatures was also investigated via carbonizing the FeOOH@PPy at 500 °C and 900 °C for comparison. SEM and TEM were employed to study the structures of Fe₃O₄@NCMTs composites in detail. When calcined at 500 °C, there is almost no production of Fe₃O₄ NPs owing to the low temperature. When the temperature was increased to 900 °C, it was found in Fig. S3(A–C)† that the resultant NPs aggregated seriously (Fig. S3(D–F)†), resulting in uneven particle sizes, and high temperature caused the collapse of some tubular structures, which were not conducive to catalytic applications.

Peroxidase-like activity of Fe₃O₄@NCMTs-700

The prepared Fe₃O₄@NCMTs-700 are expected to be efficient catalysts for peroxidase mimicking owing to their tubular structure and the synergistic effect between Fe₃O₄ NPs and the NCMTs component. Herein, we have evaluated the catalytic property of the prepared Fe₃O₄@NCMTs-700 as peroxidase mimics by employing a model catalytic reaction toward TMB substrate oxidation. As shown in Fig. 3(A–C), the color of the reaction solution of TMB, H₂O₂ and Fe₃O₄@NCMTs-700 is blue, which is due to the formation of the oxidized TMB (oxTMB) during the catalytic process. Accordingly, a typical obviously characteristic peak was observed at around 652 nm in the UV-vis spectrum for this system, which is ascribed to the formation of a radical cation (˙OH) and charge-transfer complex of TMB (TMB⁺).³³ However, no blue color change will be observed in the absence of TMB, H₂O₂ or Fe₃O₄@NCMTs-700, and no obvious absorption peak at 652 nm will be observed as well, demonstrating that Fe₃O₄@NCMTs-700 possessed excellent peroxidase-like activities.

Fig. 3 (A) Schematic diagram of the enzyme-like performance of Fe₃O₄@NCMTs-700. (B) UV-vis absorbance spectrum of varied control systems: (I) Fe₃O₄@NCMTs-700 + H₂O₂ + TMB, (II) Fe₃O₄@NCMTs-700 + H₂O₂, (III) Fe₃O₄@NCMTs-700 + TMB, (IV) H₂O₂ + TMB. (C) Visual photographs of varied control systems. Comparison of catalytic activity of different materials: (D) the related UV-absorption spectra, (E) the corresponding histogram, whose maximum value was set as 100%. (a–g represent Fe₃O₄@NCMTs-700, Fe₃O₄@NCMTs-500, Fe₃O₄@NCMTs-900, NCMTs@Fe₃O₄, Fe₃O₄ NPs, C nanotube, FeOOH@PPy, respectively). [TMB] = 0.1 mM, [H₂O₂] = 1.25 mM, and [catalyst]: 10 μg mL⁻¹.

In order to explore the synergistic catalytic behavior of Fe₃O₄@NCMTs-700, the catalytic performance of the composites was compared with those of Fe₃O₄ NPs, NCMTs nanotubes and FeOOH@PPy (Fig. 3(D and E)). As shown in Fig. 3D(a–f), Fe₃O₄@NCMTs-700 represented the highest catalytic performance. This might be attributed to the effective combination of Fe₃O₄ NPs and NCMTs, and the hollow NCMTs improve the conductivity of the material, which is conducive to the electron transfer between the reactants, thus improving the catalytic activity. Fig. 3D(a–c) demonstrated that Fe₃O₄@NCMTs-500 and Fe₃O₄@NCMTs-900 also had a certain catalytic effect; however, their enzyme-like catalytic performance is significantly weaker than that of Fe₃O₄@NCMTs-700. In addition, the catalytic activity of Fe₃O₄@NCMTs-700 was compared with that of NCMTs@Fe₃O₄Fig. 3D(d). It is worth noting that NCMTs@Fe₃O₄ is prepared by adjusting only the sequence of reaction steps without changing the amount of reaction, and Fe₃O₄ NPs are supported on the outside of NCMTs. The test results reveal that Fe₃O₄@NCMTs-700 exhibits stronger catalytic activity. This might be due to the following reasons: (1) hollow NCMTs possesses a 1D tubular structure and accessible mesopores on the NCMTs support for the enhanced mass diffusion and  transportations, thus leading to increased catalytic activity. (2) The NCMTs have a protective effect on the internally loaded Fe₃O₄ NPs, which significantly improves the stability and dispersion of the material. (3) The hollow core–shell tubular structure gives Fe₃O₄@NCMTs-700 a high specific surface area and exposes abundant active sites, thus improving the catalytic capacity.

Optimization of catalytic conditions

Since Fe₃O₄@NCMTs-700 is similar to other nanozymes and HRP, the peroxidase-like catalytic performance of Fe₃O₄@NCMTs-700 composite relies on pH values, temperature, and the concentration of nanomaterials. Thereupon, reactions with different pH (2.0–7.0), temperatures (25–55 °C), and nanomaterial concentrations (1.25–20 μg mL⁻¹) were investigated, respectively. It can be observed in Fig. 4(A and B) that the catalytic performance of the as-prepared composites increases as the pH value increases from 2.0 to 4.0, and there is a downward trend as the pH value further increases, revealing that the catalytic reaction was much easier in acidic environments. Fig. 4C displayed that the catalytic activity increased gradually when the temperature was in the range of 25 °C to 55 °C, whereas it dropped sharply when the temperature was higher than 35 °C. Accordingly, 35 °C is adopted as the optimal temperature. The impact of the concentration of Fe₃O₄@NCMTs-700 upon the catalytic activity (Fig. 4D) exhibited that the peroxidase-like performance of Fe₃O₄@NCMTs-700 increased with an increase in the dosage of Fe₃O₄@NCMTs-700, and then it remained almost constant. Therefore, the optimal concentration is 10 μg mL⁻¹.

Fig. 4 Peroxidase activities under varied conditions. (A) Varied pH values (2.0–7.0). (B) Pictures of reaction systems at different pH values. (C) Varied temperature (25–55 °C). (D) Varied amounts of Fe₃O₄@NCMTs-700 (1.25–20 μg mL⁻¹). Error bars indicate standard deviations from three repeated assays. [TMB] = 0.1 mM, [H₂O₂] = 1.25 mM. Catalyst dispersion (25 μg mL⁻¹). The error bars indicated the standard deviation of the three experiments.

Kinetics study of Fe₃O₄@NCMTs-700

In order to further explore the catalytic properties of Fe₃O₄@NCMTs-700, the steady-state kinetic assays were performed by altering the concentration of either H₂O₂ or TMB in the reaction system while retaining the other constant. The kinetic parameters, such as the maximal reaction velocity (V_max) and Michaelis–Menten constant (K_m), were achieved through the calculation using the Lineweaver–Burk plot, which is based on the Michaelis–Menten equation.³⁴

Fig. 5(A and B) shows the Michaelis–Menten curves related to the concentration of a substrate (H₂O₂ or TMB) versus the initial reaction velocity, respectively. Subsequently, the data were fitted to the double-reciprocal plots (inset of Fig. 5(A and B)), from which K_m and V_max values of Fe₃O₄@NCMTs-700 towards the substrate (H₂O₂ or TMB) were obtained. As we all know, the lower K_m value reflects a higher affinity between the enzyme and substrates, and vice versa. In this work, the K_m value of the as-prepared Fe₃O₄@NCMTs-700 for H₂O₂ and TMB are obviously inferior to other nanozymes and HRP, as shown in Table 1, suggesting a superior affinity of the nanocomposites to both substrates, which resulted from the hollow structure of Fe₃O₄@NCMTs carrying good adsorption capacity for H₂O₂ and TMB.

Fig. 5 Steady-state kinetic analysis of Fe₃O₄@NCMTs-700. (A) The TMB concentration was 0.1 mM and the concentration of H₂O₂ was varied. Inset: the corresponding double reciprocal plots of H₂O₂. (B) The concentration of TMB was varied and the H₂O₂ concentration was 1.25 mM. Inset: the corresponding double reciprocal plots of TMB. (C) The UV-vis absorption spectra with varied concentrations of H₂O (a–m: 0–2500 μM). Inset: the corresponding color changes after the reaction. (D) A dose–response curve for H₂O₂ detection in the range of 0 to 2500 μM using Fe₃O₄@NCMTs-700 as a peroxidase mimic. Inset: the linear fit of varied concentrations of H₂O₂ ranged from 2.5–75 μM. The assays were performed by adding 10 μL of Fe₃O₄@NCMTs-700 (0.4 mg mL⁻¹) in the HAc–NaAc buffer (370 μL, pH 4.0) and reacted at 35 °C for 10 min. Error bars indicate standard errors derived from three repeated measurements.

Table 1 Comparison of the apparent kinetic parameters of varied enzyme mimics and HRP

Enzyme mimics	K m (mM)		V max (10^−8 s^−1)		Ref.
	H2O2	TMB	H2O2	TMB
HRP	3.7	0.434	8.71	10	35
Fe3O4 MNPs	0.43	0.71	13.08	5.31	7
Fe3O4@C YSNs	0.035	0.27	3.34	12.0	36
Fe3O4/CoFe–LDH	10.24	0.395	—	—	37
Fe3O4@CeO2 NCs	1.13	0.15	12.5	0.64	38
GO–Fe3O4	0.71	0.43	5.31	13.08	39
NCNTs@MoS2	0.07	0.382	4.40	4.83	40
Fe3O4@NCMTs-700	0.184	0.116	5.43	10.25	This work

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Colorimetric determination of H₂O₂

Considering the excellent catalytic performance of Fe₃O₄@NCMTs-700 composites, a facile and selective method to determine H₂O₂ was developed. Fig. 5C describes the intensity of the absorbance increased with the increased concentration of H₂O₂ and the inset shows obvious color changes. In addition, a typical H₂O₂ dose–response curve is represented in Fig. 5D, and the linear regression equation was as follows: A = 0.00499[H₂O₂] (μM) + 0.13342(R² = 0.992), and the limit of detection (LOD) was 0.15 μM (S/N = 3), which represented a good linear relationship in the range of 2.5 μM to 75 μM. The obtained LOD is better than that of the previously reported determination of H₂O₂ by other nanocomposites as enzyme mimics.^41–43

Colorimetric detection for GSH

Glutathione (reduced) is regarded as a fundamental polypeptide in the human body and plays a considerable role in physiological functions.⁴⁴ Owing to the reducing ability of thiol in glutathione for blue ox-TMB into colorless TMB, an accurate and easy colorimetric method has been established and used for detecting GSH.^45–47

The colorimetric determination principle of GSH is shown in Fig. 6(A and B). Based on the peroxidase-like activity of Fe₃O₄@NCMTs-700, TMB can be oxidized to produce a blue product (oxTMB) in the presence of H₂O₂. If GSH is added, oxTMB can be rapidly reduced to TMB, followed by the fading of the blue color in the reaction solution. Moreover, the absorbance value of the reaction system at 652 nm changed obviously during the process. As depicted in Fig. 6C, the UV-vis absorbance decreases gradually as the GSH concentration increases in the range of 0–100 μM, and the inset shows the corresponding color changes. Fig. 6D indicates a concentration-response curve for GSH detection, from which the regression equation (Fig. 6D inset) was fitted as A = −0.0269C + 1.0439 (R² = 0.99599) with a wide range from 0.5 to 25 μM. The LOD of 0.49 μM (S/N = 3) was achieved through the calculation, which is far superior to some of the nanomaterial-based colorimetric approaches for GSH detection (Table 2). The obtained results demonstrated that the developed Fe₃O₄@NCMTs-700-based biosensor with high sensitivities would be feasible for detecting GSH.

Fig. 6 (A) GSH detection schematic. (B) UV-vis absorption and photograph of GSH detection. a: Without GSH, b: with GSH. (C) The UV-vis spectrum of the colorimetric sensor for GSH detection. Inset: photograph of the corresponding color changes in the solution with varied concentrations of GSH (a–l: 0–100 μM). (D) A dose–response curve for GSH detection using Fe₃O₄@NCMTs-700 as a peroxidase mimic. Inset: the linear calibration plot for GSH detection in the range of 0.5 to 25 μM. (E) Selective analysis of GSH detection (the concentrations of GSH and other interferences were 0.1 mM and 25 mM, respectively). Inset: the corresponding color changes of the solution. (ΔA = A₀ − A, A₀ and A represent the absorbance in the presence and absence of GSH, respectively). Experiments were performed by adding 10 μg mL⁻¹ of Fe₃O₄@NCMTs-700 in pH 4.0 HAc–NaAc buffer containing 0.1 mM TMB at 35 °C for 10 min. Error bars indicated the standard deviation by three independent assays.

Table 2 Comparison of different methods for the detection of GSH

Catalysts	Linear range (μM)	Detection limit (μM)	Ref.
Fe3O4 NPs	3–30	3	48
Cy–Au NCs	500–2500	30	49
NS–CQDs	20–100	4	50
Ir/NC	0.05–15	0.5	51
FeMnO3@PPy	0–10	0.36	52
Mn3O4	5–60	0.889	53
Fe3O4@NCMTs-700	0.5–25	0.49	This work

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Interference experiments

To further research the selectivity for GSH detection, the control experiments were performed using some typical common interfering components in human serum (for example, glucose, histidine, uric acid, aspartic acid and other ions such as Na⁺, K⁺, Ca²⁺ and Cl⁻). It is seen in Fig. 6E that there is a dramatical difference in absorbance at around 652 nm after adding 0.1 mM of glucose and the color variation in the reaction can also be distinguished by the naked eye (the inset of Fig. 6E). By comparison, even if the concentration of other interferences was 250 times higher than that of GSH, changes in absorbance can hardly be detected, which further indicates that the analytical method possessed good selectivity for GSH detection.

Conclusions

In summary, novel Fe₃O₄@NCMTs composites were reported via a facile and effective strategy as a highly active peroxidase-like mimetic, which exhibits a large specific surface area and abundant porous properties. It was found that Fe₃O₄@NCMTs-700 possessed excellent catalytic performance owing to the unique core–shell tubular structures with inner cavities, which could enhance nanozyme activities via an accumulation of Fe₃O₄ NPs inside. Moreover, the void space of the Fe₃O₄@NCMTs-700 is permeable for mass transfer of substrates, broadening the contact area of the substrate and the collaborative effect between the NCMTs and Fe₃O₄ NPs. In consideration of the above facts, the Fe₃O₄@NCMTs-based colorimetric biosensor was successfully exploited for GSH detection with excellent selectivity and specificity. This proposed sensing platform realized facile, economical and sensitive detection, demonstrating a tremendous potential in bioanalysis and clinical diagnosis.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grants 22174077 and 21874074).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3dt04310j

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