Bimetallic Ti₃C₂T_x with three synergistic catalytic pathways and enhanced dual enzyme activities for a visual sensing platform

Abstract

Nano-enzymes are increasingly used in forensic identification, biochemical testing, food regulation, environmental pollution monitoring and other fields. However, the construction of enzyme cascade catalytic systems based on nano-enzymes with multiple enzyme activities presents both opportunities and challenges. Ti₃AlC₂ is a common MXene with a graphene-like structure, which has the features of a large specific surface area, good electrical conductivity, excellent catalytic properties, and easy functionalization. Moreover, after being functionalized, Ti₃AlC₂ can exhibit excellent peroxidase-like activity. Therefore, in this work, a bimetallic Fe–Ni@Ti₃C₂T_x nano-enzyme with both peroxidase-like and oxidase-like activities was synthesized, and three synergistic catalytic mechanisms of Fe–Ni@Ti₃C₂T_x were verified. A colorimetric sensor was constructed based on Fe–Ni@Ti₃C₂T_x for the detection of H₂O₂ to test its feasibility for practical applications. The prepared colorimetric sensor had a wide linear range (50–6000 µM) and a low detection limit (14.606 µM). In addition, the selectivity, stability and reproducibility of the prepared colorimetric sensor were acceptable. This study laid a foundation for the simple preparation and practical application of a bimetallic nano-enzyme with various enzyme activities.

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1 Introduction

Nano-enzymes are chemically synthesized nanomaterials with natural biological enzyme activity. Compared with natural enzymes, nano-enzymes are characterized by the properties of nanomaterials, including easy synthesis, easy storage, high stability and low cost, in addition to enzyme-like activity.¹ Distinct nanomaterials, including carbon-based nanomaterials, transition metal oxides, and metals, have successfully mimicked various natural enzymes, such as peroxidase, oxidase, catalase, and superoxide dismutase, biologically.^2–4 Therefore, nano-enzymes are widely used in biochemical detection,⁵ anti-tumor therapy,⁶ environmental engineering,⁷ and food regulation.⁸ Nano-enzymes with dual activities are more conducive to practical applications. Among them, nano-enzymes with both peroxidase-like and oxidase-like activities belong to a more important category, which can skillfully constitute a cascade reaction system between the two enzyme activities, realize the self-sufficiency of the intermediate product H₂O₂ and constitute a closed-loop reaction, greatly improving the reaction efficiency.⁹

Two-dimensional layered nanomaterials (MXenes, black phosphorus, and graphitized carbon nitrides) show enormous potential application in the domain of nano-enzymes due to their large specific surface area, excellent physicochemical properties, and unique nanosheet structures.^10–12 MXene is a two-dimensional material with a graphene-like structure, consisting of transition metal carbides, nitrides or carbon-nitrides.¹³ The chemical formula is M_n+1X_nT_x, where M represents a transition metal element, X represents carbon, nitrogen and their mixture, and T_x represents a surface functional group, such as –F, –Cl, =O, or –OH.¹⁴ Due to the unique physicochemical properties, such as a large specific surface area, good electrical conductivity, excellent catalytic properties, and ease of functionalization, MXenes have been widely used in many fields, including biomedicine,¹⁵ energy storage,¹⁶ catalysis,¹⁷ and sensors.¹⁸ The special hexagonal crystal system and lamellar structure endow Ti₃AlC₂ with properties of high electric conductivity, high thermal conductivity, ease of processing, corrosion resistance, and good thermal shock resistance.¹⁹ However, the defects of easy oxidation, easy stacking between layers and poor biocompatibility of Ti₃AlC₂ limit its application. Combining Ti₃AlC₂ with nanoparticles, metal particles, and non-metallic ions can improve these drawbacks.²⁰ The functionalized Ti₃AlC₂ exhibits peroxidase-like activity, which can catalyze the production of –OH from H₂O₂ and then oxidize TMB with a color change (colorless to blue).²¹ Therefore, colorimetric sensors based on Ti₃AlC₂ are favored by researchers. Geng et al. demonstrated the synergistically enhanced catalytic activity of Ti₃C₂/Pt composites for the specific detection of glutathione and glucose based on the inhibition effect and cascade reactions, respectively.⁴ Li et al., using an in situ co-precipitation method, synthesized a MXene@NiFe-LDH nanohybrid material with strong peroxidase-like activity and successfully realized the detection of glutathione.²² Thus, functionalized MXene materials exhibit better performance.

Single-atom nano-enzymes (SA-zymes) are characterized by unique electronic structure, clear geometry, and high atom utilization efficiency.²³ Xi et al. loaded Fe atoms on Ti₃C₂T_x to form a single-atom nano-enzyme Fe–SA/Ti₃C₂T_x with excellent peroxidase-like activity, which achieved one-time recognition of five antioxidants.²⁴ Specifically, on Ti₃C₂T_x, the element Fe existed in atomic form, with one Fe atom coordinated by three oxygen atoms. The strong interaction between Fe atoms and Ti₃C₂T_x through Fe–O–Ti ligands promoted the electron transfer from Fe–SA/Ti₃C₂T_x to H₂O₂, which in turn enhanced the peroxidase-like activity. However, SA-zymes suffer from the deficiency of low loading, which can be solved by bimetallic nano-enzymes and enhance the peroxidase-like activity by the synergistic effect.²⁵ Ni is a transition metal with a small atomic radius, low electronegativity, and more valence states, in which the d-electron layer is prone to losing or capturing electrons.²⁶ Ni, owing to the advantages of its strong redox properties, stable chemical properties, and low cost, has been used to design a variety of nano-enzymes.²⁷ Therefore, the Fe–Ni bimetallic nano-enzyme may have better performance.

Based on the above discussion, in this work, Fe and Ni were loaded on Ti₃C₂T_x in a simple method for the first time. Fe–Ni@Ti₃C₂T_x exhibits strong peroxidase-like and oxidase-like activity, consuming H₂O₂ while generating H₂O₂, greatly amplifying the signal (Fig. 1).

Fig. 1 Synthesis of Fe–Ni@Ti₃C₂T_x (A) and colorimetric detection of H₂O₂ (B).

2 Experimental section

2.1 Materials

Ti₃AlC₂ was bought from Suzhou Beike Nanotechnology Co., Ltd (Jiangsu, China). Lithium fluoride (LiF), iron chloride hexahydrate (FeCl₃·6H₂O), and nickel chloride hexahydrate (NiCl₂·6H₂O) were bought from Aladdin Reagent Co., Ltd (Shanghai, China). Hydrochloric acid (HCl), TMB, and H₂O₂ were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China).

2.2 Instrumentation

Transmission electron microscopy (TEM) was performed on Tecnai G2 F20 (FEI, USA). The environment control scanning probe microscope (AFM) was from Nanonavi E-Sweep (SII, Japan). Transmission electron microscopy mapping (SEM-Mapping) was performed using QUANTA FEG 250 (FEI, USA). Energy dispersive spectroscopy (EDS) was performed using a UH5300 spectrophotometer (HITACHI, Japan). X-ray diffraction (XRD) was carried out using D8 ADVANCE X (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was performed by Axis Ultra DLD (Kratos, England). The absorption spectra were recorded on a TU-1901 ultraviolet (UV) spectrophotometer (Purkinje, China). Ultra-pure water was obtained by Milli-Q purification system (Merck Millipore, Germany), which was used throughout the entire experiment.

2.3 Synthesis of Ti₃C₂T_x

Ti₃AlC₂ was synthesized according to a reported method with slight modifications.²⁴ LiF (3.2 g) was added to HCl (12 M 40 mL) and heated to 45 °C with stirring for 10 min. Ti₃AlC₂ (2 g) was added to the above mixed solution and stirred magnetically at 45 °C for 48 h. The solution was then poured into a centrifuge tube and centrifuged for 5 min. The above solution was rinsed three times with dilute HCl (2 M), followed by several rinses with ultra-pure water until the pH of the upper layer was close to 6 (centrifugation at 3500 rpm for 5 min was performed to ensure a thorough separation of the upper liquid from the precipitate). Next, the mixture was poured into a gas collection flask and sonicated under nitrogen and ice bath conditions for 1 h. Finally, the mixture was poured into a centrifuge tube and centrifuged for 45 min. The upper layer of the black liquid comprised single/few-layer Ti₃C₂T_x nanosheets, which were sealed and stored in the refrigerator at 4 °C for use.

2.4 Synthesis of Fe–Ni@Ti₃C₂T_x

The fabrication of Fe–Ni@Ti₃C₂T_x was carried out with slight modifications on the basis of an existing study.²⁴ FeCl₃·6H₂O (6 mg) and NiCl₂·6H₂O (9 mg) were added to Ti₃C₂T_x (10 mL), stirred for 10 min and sonicated for 30 min to obtain Fe–Ni@Ti₃C₂T_x peroxidase, and the synthetic material was then preserved in a lightproof refrigerator (4 °C) (Fig. 1A).

2.5 Preparation of the sensor

The preparation was completed by first adding H₂O₂ (0.5 mL), then TMB (3.5 mL) and finally Fe–Ni@Ti₃C₂T_x (1 mL) to an empty test tube. Measurements were carried out under optimum experimental conditions (50 °C, 13 min, pH = 3) using a UV spectrophotometer, as shown in Fig. 1B.

3 Results and discussion

3.1 Optimization and characterization of synthesized materials

3.1.1 The quality optimization of FeCl₃·6H₂O and NiCl₂·6H₂O. The modulation of the Fe/Ni ratio tailors the electronic configuration of bimetallic active sites, including charge-density redistribution and interfacial bonding modes between Fe and Ni, thereby reinforcing the synergistic catalytic efficacy.²⁸ The balanced Fe/Ni proportion can also optimize the enzymatic-like reaction kinetics and ameliorate the structural stability of the composite, which enhances its anti-interference capability.²⁹ Keeping the experimental conditions unchanged (0.5 mL 100 µM H₂O₂, 3.5 mL TMB, 1 mL Fe–Ni@Ti₃C₂T_x, 50 °C, 13 min, pH = 3), the Fe and Ni contents in Fe–Ni@Ti₃C₂T_x were optimized. FeCl₃·6H₂O (1, 3, 6, 9, 12 mg) and 9 mg NiCl₂·6H₂O were added to 10 mL of Ti₃C₂T_x, respectively, with stirring for 10 min and ultrasonication for 30 min. It can be noted from Fig. S1A that the strongest absorption peak and the highest absorbance were obtained when FeCl₃·6H₂O was 6 mg per 10 mL of Ti₃C₂T_x. Under the same experimental conditions, NiCl₂·6H₂O (1, 3, 6, 9, 12 mg) and 6 mg FeCl₃·6H₂O were added to five 10 mL Ti₃C₂T_x, stirred for 10 min, and ultrasonicated for 30 min. The optimum absorption peak and the highest absorbance were obtained with 9 mg of NiCl₂·6H₂O. In brief, with 6 mg FeCl₃·6H₂O and 9 mg NiCl₂·6H₂O per 10 mL of Ti₃C₂T_x, the strongest absorption peak can be obtained and Fe–Ni@Ti₃C₂T_x exhibits the highest peroxidase activity (Fig. S1B).

3.1.2 Characterization of Ti₃C₂T_x and Fe–Ni@Ti₃C₂T_x. The morphology of Ti₃C₂T_x was characterized by TEM and AFM. As shown in Fig. 2A(a and b), the prepared Ti₃C₂T_x was composed of layered nanosheets. AFM images revealed that the surface of Ti₃C₂T_x exhibited nanoscale roughness, with a maximum peak-to-valley height difference of approximately 7.1 nm and an average surface fluctuation of about 1.9 nm (Fig. 2A(c)). The surface roughness of Ti₃C₂T_x contributes to enhanced metal adsorption capacity, thereby improving its catalytic performance. XRD and XPS data were in agreement with those reported in the study, which further confirmed the successful synthesis of single/few-layer Ti₃C₂T_x nanosheets as shown in Fig. S3A and C.²⁴ As illustrated in Fig. 2B(a and b), the TEM of Fe–Ni@Ti₃C₂T_x showed that Fe–Ni@Ti₃C₂T_x retained a good nanosheet structure, and Fe–Ni nanoparticles were observed to be uniformly dispersed on Ti₃C₂T_x nanosheets. Fig. 2B(c) shows Fe nanoparticles on the Ti₃C₂T_x surface, while Fig. 2B(d) shows Ni nanoparticles on the Ti₃C₂T_x surface. Fig. 2B(e and f) confirms the uniform distribution of Fe and Ni nanoparticles across the Ti₃C₂T_x surface, with average particle sizes of 4.000 nm for Fe and 1.123 nm for Ni. As shown in Fig. 2C(a–c), a large number of spots in the aberration-corrected HAADF-STEM (AC-HAADF-STEM) indicated the presence of Fe and Ni nanoparticles on Fe–Ni@Ti₃C₂T_x. The lattice fringes observed in the images correspond to the (110) crystal plane of Fe. The EDS mapping demonstrated that the elements of C, Ti, O, Fe, and Ni were homogeneously distributed on Fe–Ni@Ti₃C₂T_x (Fig. S2). In addition, the elemental composition of the Fe–Ni@Ti₃C₂T_x surface and its chemical state were analyzed by XRD and XPS, as shown in Fig. S3B and D. XRD showed diffraction peaks related to Fe²⁺ and Ni²⁺ compounds. XPS showed a distinct peak at 710.4 eV, corresponding to Fe 2p, and at 855.6 eV, corresponding to Ni 2p, which further proved the presence of Fe²⁺ and Ni²⁺ compounds in Ni@Ti₃C₂T_x.^30,31

Fig. 2 (A) Characterization of Ti₃C₂T_x. TEM (a and b) and AFM images (c). (B) Characterization of Fe–Ni@Ti₃C₂T_x. TEM images of Fe–Ni@Ti₃C₂T_x (a and b), Fe nanoparticles (c) and Ni nanoparticles (d) and size distribution of Fe nanoparticles (e) and Ni nanoparticles (f). (C) AC HAADF-STEM image of Fe–Ni@Ti₃C₂T_x. With a scale bar of 5 nm (a) and 2 nm (b), and (c) magnified view of the cropped region from (b).

3.2 The performance advantages of Fe–Ni@Ti₃C₂T_x

3.2.1 Verification of dual enzyme activities of Fe–Ni@Ti₃C₂T_x. Based on the following principles: (1) peroxidase-like activity can change TMB from colorless to blue oxTMB in the TMB/H₂O₂ system; (2) oxidase-like activity can directly change TMB from colorless to blue oxTMB; (3) a strong absorption peak at 650 nm can be observed when Fe–Ni@Ti₃C₂T_x has peroxidase-like or oxidase-like activities. The following sets of experiments were designed to validate the dual enzyme activities of Fe–Ni@Ti₃C₂T_x: (1) H₂O₂ + Fe–Ni@Ti₃C₂T_x; (2) TMB + H₂O₂; (3) TMB + Fe–Ni@Ti₃C₂T_x; and (4) TMB + H₂O₂ + Fe–Ni@Ti₃C₂T_x under optimal experimental conditions (0.5 mL 100 µM H₂O₂, 3.5 mL TMB, 1 mL Fe–Ni@Ti₃C₂T_x/ultra-pure water, 50 °C, 13 min, pH = 3). The UV-vis adsorption spectra of different reaction systems are shown in Fig. S4. After the addition of Fe–Ni@Ti₃C₂T_x to the TMB/H₂O₂ system, colorless TMB turned into blue oxTMB, and the absorption peak at 650 nm in the UV-vis spectrum appeared much higher than that of other experimental groups, indicating that Fe–Ni@Ti₃C₂T_x has strong peroxidase-like activity. The TMB + Fe–Ni@Ti₃C₂T_x system also showed the absorption peak at 650 nm, but with a lower intensity than that of the TMB + H₂O₂ + Fe–Ni@Ti₃C₂T_x system, suggesting that Fe–Ni@Ti₃C₂T_x had oxidase-like activity, which can catalyze the oxidation of TMB by O₂. Meanwhile, a slight color change and absorption peak were observed in the TMB + H₂O₂ system, indicating the oxidizing property of H₂O₂. The above results confirmed that Fe–Ni@Ti₃C₂T_x possessed strong peroxidase-like and oxidase-like catalytic activity.

3.2.2 The enzyme activity of Fe–Ni@Ti₃C₂T_x is superior to that of Ti₃C₂T_x, Fe@Ti₃C₂T_x and Ni@Ti₃C₂T_x. Ni@Ti₃C₂T_x was synthesized by adding 9 mg NiCl₂·6H₂O to 10 mL Ti₃C₂T_x, stirring for 10 min and sonicating for 30 min. Fe@Ti₃C₂T_x was synthesized by mixing 6 mg FeCl₃·6H₂O and 10 mL Ti₃C₂T_x, stirring for 10 min and sonicating for 30 min. 1 mL of Ti₃C₂T_x, Ni@Ti₃C₂T_x, Fe@Ti₃C₂T_x, and Fe–Ni@Ti₃C₂T_x were added to three test tubes containing 0.5 mL 100 µM of H₂O₂ and 3.5 mL of TMB, respectively, and under the optimal conditions (50 °C, 13 min, pH = 3), the absorbances were detected and the absorption peaks at 650 nm were compared. As demonstrated in Fig. S5, the H₂O₂ +TMB + Ti₃C₂T_x, H₂O₂ +TMB + Ni@Ti₃C₂T_x, H₂O₂ +TMB + Fe@Ti₃C₂T_x, and H₂O₂ +TMB + Fe–Ni@Ti₃C₂T_x systems all showed an obvious absorption peak at 650 nm in the UV-vis spectrogram, but the absorbance and absorption peak at 650 nm of the H₂O₂ + TMB + Fe–Ni@Ti₃C₂T_x system were much higher than those of the Ti₃C₂T_x, Ni@Ti₃C₂T_x and Fe@Ti₃C₂T_x systems, suggesting that the enzyme activity of Fe–Ni@Ti₃C₂T_x was superior than those of Ti₃C₂T_x, Ni@Ti₃C₂T_x and Fe@Ti₃C₂T_x, with Fe–Ni playing a synergistic catalytic role.

3.2.3 The enzyme activity of Fe–Ni@Ti₃C₂T_x is significantly better than that of HRP. Under the optimal experimental conditions, the absorbance of 9 groups of 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL Fe–Ni@Ti₃C₂T_x system and 9 groups of 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL HRP system were measured using a UV-vis spectrophotometer. The absorbance (Fig. S6A) and the absorption peak at 650 nm of 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL Fe–Ni@Ti₃C₂T_x system (Fig. S6B) were significantly higher than that of the 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL HRP system. Comparison of enzyme activity between Fe–Ni@Ti₃C₂T_x and HRP was performed using a t-test of two independent samples, which resulted in P < 0.05, verifying that the enzyme activity of Fe–Ni@Ti₃C₂T_x was much stronger than that of HRP.

3.3 Catalytic mechanism of Fe–Ni@Ti₃C₂T_x

The catalytic mechanism of Fe–Ni@Ti₃C₂T_x to the H₂O₂/TMB system is presented in Fig. 3. Fe²⁺ can undergo a Fenton reaction with H₂O₂ to produce –OH. Meanwhile, Fe atoms can interact with Ti₃C₂T_x through the Fe–O–Ti ligand to improve the electron transfer ability and catalytic ability, which accelerated the decomposition of H₂O₂ and produced a large amount of –OH.⁶ The addition of Ni not only improved the low loading, but also synergized the catalytic reaction and increased the yield of –OH, which further improved the catalytic activity.³² Therefore, a large quantity of –OH oxidized TMB to produce H₂O and oxTMB, and the solution changed from colorless to blue. In addition, Fe–Ni@Ti₃C₂T_x is a nano-enzyme with both peroxidase-like and oxidase-like activities, which can combine the ability of oxidase to reduce O₂ to H₂O₂, with the peroxidase to consume H₂O₂ and produce –OH, forming a cascade enzyme-catalyzed system that amplifies the reaction signal.

Fig. 3 Catalytic mechanism of Fe–Ni@Ti₃C₂T_x.

3.4 Optimization of detection conditions

3.4.1 Optimization of the volume ratio of TMB and Fe–Ni@Ti₃C₂T_x. The content of TMB and Fe–Ni@Ti₃C₂T_x had a significant effect on absorbance. In order to control the concentration of H₂O₂, the total volume of the H₂O₂ + TMB + Fe–Ni@Ti₃C₂T_x system was set at 5 mL, and the volume of H₂O₂ was fixed to be 0.5 mL with a concentration of 100 µM. In order to obtain the best volume ratio, the following sets of experiments were designed, including 0.5 mL H₂O₂ + 2 mL TMB + 2.5 mL Fe–Ni@Ti₃C₂T_x, 0.5 mL H₂O₂ + 2.5 mL TMB + 2 mL Fe–Ni@Ti₃C₂T_x, 0.5 mL H₂O₂ + 3 mL TMB + 1.5 mL Fe–Ni@Ti₃C₂T_x, 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL Fe–Ni@Ti₃C₂T_x, and 0.5 mL H₂O₂ + 4 mL TMB + 0.5 mL Fe–Ni@Ti₃C₂T_x. As depicted in Fig. S7, under optimal experimental conditions (50 °C, 13 min, pH = 3), the highest absorbance was obtained with the 0.5 mL H₂O₂ + 3.5 mL TMB + 1 mL Fe–Ni@Ti₃C₂T_x system.

3.4.2 Optimization of pH, time, and temperature. The pH, time, and temperature may affect the dual enzyme activities of Fe–Ni@Ti₃C₂T_x.³³ The reaction time was defined as the mixing time of Fe–Ni@Ti₃C₂T_x in the H₂O₂/TMB system. In order to confirm the optimal experimental conditions, the absorbance of the system was investigated at different pH (2–6), reaction times (11–15 min) and temperatures (20–60 °C). As illustrated in Fig. S8, pH, reaction time and temperature all had a large effect on the dual enzyme activities of Fe–Ni@Ti₃C₂T_x. With the progressively increasing pH, reaction time and temperature, the dual enzyme activities of Fe–Ni@Ti₃C₂T_x initially increased and then decreased. The results demonstrated that the optimal values of pH, reaction time and temperature were 3, 13 min, and 50 °C, respectively.

3.5 Colorimetric detection of H₂O₂

A colorimetric sensor was constructed based on Fe–Ni@Ti₃C₂T_x for the detection of H₂O₂ to test the feasibility of Fe–Ni@Ti₃C₂T_x in practical applications. The UV-vis absorption spectra were plotted to characterize the colorimetric sensor assembly under optimal conditions (0.5 mL 100 µM H₂O₂, 3.5 mL TMB, 1 mL Fe–Ni@Ti₃C₂T_x, 50 °C, 13 min, pH = 3). As shown in Fig. S9, the absorption spectrum of H₂O₂ tends to 0, and no characteristic absorption peak was observed. After the addition of TMB, a smaller absorption peak appeared at 650 nm. This was attributed to the strong oxidizing properties of H₂O₂, which can decompose a small amount of –OH to oxidize TMB even without catalysis by peroxidase. Finally, by adding Fe–Ni@Ti₃C₂T_x, the absorption peak at 650 nm increased significantly due to the excellent dual enzyme activities of Fe–Ni@Ti₃C₂T_x, which can consume O₂ to produce H₂O₂ and catalyze the production of large amounts of –OH from H₂O₂ to oxidize TMB. The above conclusions indicated that the prepared colorimetric sensor achieved the expected results. The impact of H₂O₂ concentration on the absorbance at 650 nm under the optimal experimental conditions is presented in Fig. 4A. In the range of 50–6000 µM, the absorbance gradually increased with the increase in H₂O₂ concentration. The absorbance of the H₂O₂ + TMB + Fe–Ni@Ti₃C₂T_x system showed a good linear relationship with the concentration of H₂O₂, with the linear equation y = 0.000118582x + 3.84262 and detection limit 14.606 µM (3σ/k) (Fig. 4B). Table. S1 compares the results of various reports for the detection of H₂O₂.^34–40

Fig. 4 (A) UV–vis absorption spectra of the colorimetric sensor with different concentrations of H₂O₂ (50–6000 µM). (B) Linear calibration plot of H₂O₂ detection.

The detection materials and their ratios employed in different experiments can influence the enzyme-like activity of MXene-based materials. The structural and compositional diversity of MXene materials (Ti₃C₂T_x, Nb₂CT_x, and Mo₂CT_x) confers superior catalytic performance when hybridized with heterogeneous components, thereby expanding their applicability across biomedicine,⁴¹ electronic engineering,⁴² environmental remediation,⁴³ and renewable energy sectors.⁴⁴ Specifically, the CdS/Ti₃C₂ heterojunction system facilitates multi-electron reduction pathways, enabling efficient H₂ evolution catalysis.⁴⁵ Nitrogen-doped Ti₃C₂ quantum dots enable sensitive H₂O₂ detection through catalytic photoinduced electron transfer mechanisms.⁴⁶ MXene-supported metal phosphide composites (NiCoP@Ti₃C₂T_x) exhibit dual-functional catalytic behavior: under alkaline conditions, they mediate OH⁻ adsorption and subsequent H₂O formation, while under acidic environments, they promote H⁺ reduction—both processes synergistically accelerating the hydrogen evolution reaction.⁴⁷ This study selected the most commonly used Ti₃C₂T_x, whose physicochemical properties are well established. Its combination with bimetallic components facilitated a more effective investigation of enzyme-mimetic properties, thereby enhancing the credibility of the results. Compared to other studies utilizing MXene-based materials for H₂O₂ detection, the present approach offered several advantages, including a detection mechanism that enables triple synergistic effects, a stable detection system, convenient operation, and low cost. In conclusion, the colorimetric sensor based on Fe–Ni@Ti₃C₂T_x showed a wide linear range and a low detection limit due to its peroxidase-like and oxidase-like activities, which provided a simple method for the detection of H₂O₂.

3.6 Selectivity, stability and reproducibility tests

The specificity of the colorimetric sensor for H₂O₂ was assessed by comparing the absorbance of the sensor in the presence or absence of interfering substances. K⁺, Na⁺, Al³⁺, Cr³⁺, Li⁺, Ca²⁺, Mn²⁺, F⁻, HPO₄²⁻, CO₃²⁻, and NO₃⁻ were selected as potential interfering substances, with concentrations nearly 100-fold higher than that of H₂O₂. As presented in Fig. 5A, except for CO₃²⁻ and NO₃⁻, other interferences had no significant effect on the detection of H₂O₂, which may be attributed to the strong oxidation of H₂O₂, and both CO₃²⁻ and NO₃⁻ undergo redox reactions with H₂O₂. In other words, they would not coexist in a real environment. Therefore, the specificity of the colorimetric sensor is acceptable.

Fig. 5 (A) Absorbance of the reaction system toward interfering species. (B) Absorbance of the reaction system after 0, 1, 2, and 3 weeks. (C) Absorbance of the reaction system under the same conditions in 9 groups.

In practice, the stability of the catalyst is an important factor that affects the whole reaction system. The stability of this colorimetric sensor was examined by storing Fe–Ni@Ti₃C₂T_x at 4 °C for 0, 1, 2 and 3 weeks. As revealed in Fig. 5B, the peroxidase activity of Fe–Ni@Ti₃C₂T_x to various concentrations of H₂O₂ (100 µM, 1000 µM, and 3000 µM) remained basically unchanged, indicating that the colorimetric sensor based on Fe–Ni@Ti₃C₂T_x for the detection of H₂O₂ showed good stability.

In order to evaluate the reproducibility of this colorimetric sensor, the same nine sets of systems were prepared for simultaneous detection with 100 µM, 1000 µM, and 3000 µM H₂O₂. As presented in Fig. 5C, the relative standard deviation (RSD) for 100 µM, 1000 µM, and 3000 µM H₂O₂ was 5.64%, 2.67%, and 2.95%, respectively, suggesting that this colorimetric sensor had a good reproducibility.

3.7 Detection of real samples

In order to evaluate the applicability of this colorimetric sensor for the detection of H₂O₂ in real samples, the recovery of H₂O₂ was analyzed using both blank spiked recovery and sample spiked recovery. The recoveries for 500 µM, 1000 µM, and 3000 µM H₂O₂ were 106.37%, 96.39%, and 101.08%, with RSDs of 2.63%, 1.15%, and 1.65%, respectively (Table 1). After centrifuging the milk purchased from a local supermarket for 2 h, 500 µM, 1000 µM, and 3000 µM of H₂O₂ were added, and the recoveries were 104.31%, 102.59% and 100.92%, with RSDs of 4.99%, 4.54% and 3.38%, respectively, which indicated that the colorimetric sensor was suitable for the detection of H₂O₂ in real samples.

Table 1 Determination of H₂O₂ in milk using the as-prepared colorimetric sensor

Type	Add (µM)	Sample 1	Sample 2	Sample 3	Found (µM)	Recovery (%)	RSD (%)
Blank	500	543	536	516	531.87	106.37%	2.63%
	1000	952	974	965	963.90	96.39%	1.15%
	3000	2977	3044	3075	3032.41	101.08%	1.65%
Milk	500	547	522	495	521.53	104.31%	4.99%
	1000	986	1014	1077	1025.91	102.59%	4.54%
	3000	3036	3125	2921	3027.75	100.92%	3.38%

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4 Conclusion

In summary, a novel bimetallic artificial nano-enzyme Fe–Ni@Ti₃C₂T_x with peroxidase-like or oxidase-like activities based on Ti₃C₂T_x was prepared via a simple method. Fe–Ni@Ti₃C₂T_x can catalyze the production of –OH from H₂O₂ by multiple mechanisms, and then oxidize the colorless TMB to the colored oxTMB. A sensitive, efficient, convenient and economical colorimetric sensor based on Fe–Ni@Ti₃C₂T_x was constructed for the detection of H₂O₂ to validate the dual enzyme activities and practical applicability of Fe–Ni@Ti₃C₂T_x, laying the groundwork for the application of bimetallic nano-enzymes with multiple enzymatic activities.

Author contributions

Zhiren Zhou: data curation, formal analysis, investigation, software, visualization, writing-original draft. Lina Zou: formal analysis, investigation, methodology, project administration, software, writing-original draft. Ping Zhang: data curation, formal analysis, investigation. Jing Dong: data curation, formal analysis, investigation. Jian Zhou: data curation, formal analysis, investigation. Hao Jiang: data curation, formal analysis, investigation. Huanyu Ren: data curation, formal analysis, investigation. Zheng Li: data curation. Huiru Niu: data curation. Hao Liao: data curation. Xiaojing Zhang: data curation. Shanshan An: data curation. Fei Ren: data curation. Xiuhong Ge: data curation. Lang Cheng: data curation. Feiyan Yang: data curation. Hongzhi Pan: methodology. Shengzhong Rong: methodology, project administration, supervision, validation, writing-review & editing. Hongkun Ma: methodology, project administration.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

The data that support the findings of this study are available from the corresponding author on request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d5na00939a.

Acknowledgements

This research was financially supported by the Fundamental Research Funds for the Universities of Heilongjiang Province (2024-KYYWF-0455).

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Footnote

† These authors contributed equally to this work.

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