Bimetallic Fe₃O₄@Co₃O₄/CN as a Nanozyme with Dual Enzyme-Mimic Activities for the Colorimetric Determination of Mercury(II)

Abstract

Colorimetric biosensor-based nanozymes have received considerable attention in various fields thanks to the advantages of the simple preparation, good stability, and regulable catalytic activity of nanozymes. In this study, a bimetallic nanozyme Fe₃O₄@Co₃O₄/CN was prepared via the high-temperature calcination of Fe₃O₄-PVP@ZIF-67. The material retained its skeletal structure before calcination, which prevented the aggregation of nanoparticles and exposed more active sites of the nanozyme, substantially enhancing the intrinsic dual enzyme-mimetic activities, including peroxidase- and oxidase-like activities. In particular, Fe₃O₄@Co₃O₄/CN with oxidase-like activity catalyzed the colorless tetramethylbenzidine (TMB) to become blue oxTMB with oxygen. Reducing glutathione (GSH) could inhibit the above oxidation reaction. In contrast, with respect to the existence of mercury(II), GSH bound to mercury(II) due to the strong affinity between mercury(II) and -SH, thus eliminating the inhibition and restoring the oxTMB signal. A simple and effective colorimetric sensor was fabricated to detect mercury(II) based on the above principles. The proposed measurement had a linear range of 0.1–15 μM and a limit of detection (LOD) of 0.017 μM. It was shown that the established colorimetric sensing system could be successfully applied to detect mercury(II) in water samples, and the Fe₃O₄@Co₃O₄/CN nanozyme proved to be a promising candidate for biosensing application.

1. Introduction

Mercury is one of the common heavy metals that pollute the environment, and it can enter living organisms through the food chain [1]. Moreover, what is worse is that the mercury ion (Hg²⁺) has a long half-life and tends to accumulate in the human body. Even low concentrations of Hg²⁺ can cause serious damage to humans by altering the structure of protein molecules, breaking hydrogen bonds, and inhibiting enzyme activity [2,3,4]. Therefore, it is of great importance to develop a sensitive, fast, and simple method for the detection of mercury. Thus far, the main methods for the detection of mercury were atomic absorption spectrometry [5], inductively coupled plasma mass spectrometry [6], inductively coupled plasma optical emission spectrometry [7], electrochemiluminescence analysis [8], fluorescence [9], and colorimetric methods [10,11,12]. Compared with the detection method with the help of expensive and complex instruments, the colorimetric method was favored by researchers for its simplicity and having no need for professional operation, as well as its instant detection, fast detection speed, and low cost.

In particular, a colorimetric sensor for Hg²⁺ detection based on natural enzyme-like horseradish peroxidase (HRP) with high catalytic activity and specificity has aroused substantial attention due to its simplicity and novelty [13]. However, natural enzymes also have some shortcomings, such as poor stability, high costs, and difficult recovery, which also hinder their application. Therefore, in order to overcome these difficulties, some nanozymes that can replace natural enzymes came into being. Nanozymes are a class of nanomaterials with properties similar to natural enzymes, with high catalytic efficiencies, low costs, easy preparations, high stabilities, easy modifications and functionalizations, etc. The emergence of nanozymes compensated for the lack of natural enzymes and broadened the application of colorimetric biosensing. In recent years, many nanozymes, including noble metals [14], metal oxides [15], carbon-based nanomaterials [16], polymers [17], and metal–organic frameworks [18], have been reported to have intrinsic nanozyme activities (single peroxidase-like activity and dual or multiple enzyme-mimetic activities for colorimetric biosensing applications). For example, FeNPs@Co₃O₄ [19] and Fe₃O₄@NH₂-MIL-100(Fe) [20] with peroxidase-like activities were applied to detect glucose. The Fe₃O₄@MIL-100(Fe) prepared using a microwave-assisted method showed satisfactory dual peroxidase-like and catalase-like activities and was used for glutathione and cholesterol sensing [21,22]. Fe₃O₄@C@MnO₂ derived from Fe-MIL-88A was reported to have triple enzyme-mimetic activities (including peroxidase-, oxidase-, and catalase-like activity) and was applied for the colorimetric biosensing of dopamine [23]. Through the above description, the development of nanozymes for biosensing was significantly practical and forward-looking.

The colorimetric strategy of heavy metal detection based on nanozymes also had broad application prospects. For example, CS/Cu/Fe [24], CoS [25], CS-SeNPs [26], MnO₂ [27], and Fe₃O₄@ZnO [28] as nanozymes were all utilized to detect mercury ions, and this was grounded in the restoration of the oxidation of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) by nanozymes and the detection of Hg²⁺ through the inhibition of glutathione (GSH) or cysteine (Cys). Specifically, thanks to their unique coordination structure and excellent properties, metal–organic frameworks (MOFs) and their derivatives have also been widely exploited as nanozymes for mercury ion colorimetric biosensing. For instance, Fe₃O₄@ZIF-67 [29], Fe-N/S-C [30], Co/Zn-MSN [31], and hollow MnFeO [32] nanozymes were used to establish novel colorimetric transport platforms for the detection of mercury ion. This is largely based on the fact that nanozymes’activity could be inhibited by Cys or GSH, and the inhibition could be released by Hg²⁺ due to the affinity of Cys or GSH for the mercury ion.

Nevertheless, MOF-derived magnetic bimetallic composite materials have rarely been reported for the colorimetric sensor detection of Hg²⁺ based on enzyme-mimetic activity.

The magnetic bimetallic oxides derived from MOFs synthesized using a microwave method have not only enhanced enzyme-like activity but also the characteristic of easy separation, making them a powerful tool for develo** colorimetric sensors [15]. In 2021, our group first synthesized Fe₃O₄-PVP@ZIF-67 within 30 min using a microwave-assisted method [33]. Later, it was demonstrated to have peroxidase-like activity, and a colorimetric sensing method for detecting the mercury ion was further developed based on the Fe₃O₄-PVP@ZIF-67 and in the presence of H₂O₂. However, this colorimetric method still needed the unstable explosive reagent H₂O₂; Fe₃O₄-PVP@ZIF-67 does not have multiple enzyme-mimetic activities, and the peroxidase-like activities needed to be further improved. We expect that bimetallic derivatives obtained via the calcination of Fe₃O₄-PVP@ZIF-67 would exhibit enhanced dual enzyme-mimetic activities for colorimetric Hg²⁺ sensing sensitivity, and this can be quickly carried out without H₂O₂.

Herein, the derivative Fe₃O₄@Co₃O₄/CN was first prepared via the high-temperature calcination of Fe₃O₄-PVP@ZIF-67 synthesized via a microwave-assisted method. The obtained Fe₃O₄@Co₃O₄/CN showed satisfactory intrinsic dual enzyme-mimetic activities, including peroxidase- and oxidase-like activities and excellent magnetic separation properties. In particular, the oxidase activity of Fe₃O₄@Co₃O₄/CN under different reaction conditions was investigated in detail. The Fe₃O₄@Co₃O₄/CN with oxidase-like activity catalyzes the oxidation reaction between colorless TMB and oxygen in the air to produce blue-green oxidation products (oxTMB). When GSH was present in the system, the reducing substance GSH inhibited the production of oxTMB from the TMB catalyzed by Fe₃O₄@Co₃O₄/CN. However, when both Hg²⁺ and GSH were present in the reaction system, the process of Fe₃O₄@Co₃O₄/CN-catalyzed TMB producing oxTMB can proceed normally due to the strong affinity of Hg²⁺ and GSH to form a coordination bond, which can release the inhibition of GSH. Based on this mechanism, a sensitive and simple colorimetric sensing method for the detection of Hg²⁺ was established under optimal experimental conditions. The colorimetric method was demonstrated to be sensitive and was applied to detect Hg²⁺ in water samples.

2. Materials and Methods

2.1. Reagents and Materials

Ethylene glycol, ethanol (EtOH), acetic acid (HAc), sodium acetate (NaAc), hydrogen peroxide (30%, H₂O₂), ferric chloride hexahydrate (FeCl₃·6H₂O), and sodium hydroxide (NaOH) were acquired from Hengxing Chemical Reagent Manufacturing Co., Ltd. (Shanghai, China). Cobaltous nitrate hexahydrate (Co(NO₃)₂·6H₂O) and polyvinyl pyrrolidone (PVP) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Glutathione (GSH), 2-methylimidazole (2-MIM), and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) were purchased at Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Thiourea(CH₄N₂S), p-Benzoquinone(C₆H₄O₂), and sodium azide(NaN₃) were taken from Bodi Chemical Co., Ltd. (Tian**, China). The mercury standard solution (1000 μg·mL⁻¹) was purchased from Bei**g North Weiye Institute of Metrology. Ultrapure water was produced by the SZ-93A auto-redistilled water system (Shanghai, China). All reagents were at least of analytical grade and were used without further purification.

2.2. Apparatus and Characterization

A UV-5100 spectrophotometer (Metash, Shanghai, China) was used to obtain the UV–vis spectrum and absorbance. The EQUINOX 55 spectrometer (Bruker, Germany) was used for the measurement of FTIR spectra. The morphology of synthesized materials was recorded on a GeminiSEM 300-71-31 field emission scanning electron microscope (Carl Zeiss, Jena, Germany). X-ray diffraction (XRD) measurements were carried out on a Smart Lab 9 kW diffractometer (Rigaku Corporation, Akishima, Japan) with 5°/min. The magnetic properties of the prepared magnetic materials were assessed using a LakeShore 7404 vibrating sample magnetometer (VSM) (LakeShore, Carson, CA, USA).

2.3. Synthesis of Fe₃O₄@Co₃O₄/CN

Firstly, Fe₃O₄-PVP@ZIF-67 was synthesized via the microwave method with reference to previous works in the laboratory [33] (the synthesis process is shown in Supplementary Materials (SM) Text S1). Secondly, Fe₃O₄@Co₃O₄/CN derivatives were prepared via the high-temperature calcination of Fe₃O₄-PVP@ZIF-67, and the ZIFs were directly carbonized by a tube furnace without the addition of additional carbon and nitrogen sources, and high-temperature carbonization could be converted into a highly nitrogen-doped carbon substrate to improve the catalytic activity of nanozymes. Finally, Fe₃O₄@Co₃O₄/CN composites with dual enzyme-mimetic activities were obtained. The process was as follows: the Fe₃O₄-PVP@ZIF-67 powders placed in a porcelain ark were calcined at 800 °C for 3 h in a tube furnace with a heating rate of 10 °C/min under a nitrogen atmosphere. The synthesis diagram of the materials is shown in Scheme 1.

2.4. Dual Enzyme-like Activities of Fe₃O₄@Co₃O₄/CN

A series of experiments were conducted to study the dual enzyme-like activity of Fe₃O₄@Co₃O₄/CN. TMB was used as the substrate in the presence or absence of H₂O₂ to verify the peroxidase-like activity and oxidase-like activity of Fe₃O₄@Co₃O₄/CN, respectively. For peroxidase-like activity, the material can catalyze the H₂O₂ oxidation of colorless TMB to produce a blue-green oxidation product (oxTMB) with maximum absorption at 652 nm, similar to horseradish peroxidase. For the oxidase-like case, the material can directly catalyze the oxygen oxidation of TMB to oxTMB without H₂O₂. Several factors affecting the activities of nanozymes, such as pH, the concentration of the material and H₂O₂, reaction times, and reaction temperatures, were further investigated. The specific procedures of dual enzyme-like catalytic performances for Fe₃O₄@Co₃O₄/CN are shown in Supplementary Materials (SM), Text S3.

To explore the catalytic activity of nanozymes, a kinetic study of the enzymatic behavior of nanozymes was conducted. Under the optimal reaction conditions, the concentration of the TMB substrate was changed, and the absorbance at 652 nm was measured without any change in other conditions. The maximum reaction rate (V_max) and Mie’s constant (K_m) were calculated according to a typical Michaelis–Menten kinetic model: $\frac{1}{V}=\frac{k_m}{V_{\mathit{max}}}(\frac{1}{\left[S\right]}+\frac{1}{K_m})$ where V_max is the maximum reaction rate, and [S] is the TMB substrate’s concentration.

In order to investigate the catalytic mechanism of nanozymes, various free radical quenchers (thiourea, p-benzoquinone, and NaN₃) were added to the reaction system at different concentrations. In order to further investigate the catalytic mechanism of the nanozymes in depth, the role of dissolved oxygen in catalysis was explored using N₂ and O₂ relative to the reverse system. The above reactions were carried out under optimal reaction conditions.

2.5. Application of Fe₃O₄@Co₃O₄/CN as Oxidase Mimics for Mercury Ion Sensing

As an oxidase mimic, Fe₃O₄@Co₃O₄/CN can catalyze the oxygen oxidation of colorless TMB to blue oxTMB, with maximum absorption at 652 nm. Reducing glutathione (GSH) could inhibit the above oxidation reaction. In contrast, with respect to the existence of mercury(II), GSH bound to mercury(II) due to the strong affinity between mercury(II) and -SH, thus eliminating the inhibition and restoring the oxTMB signal. A convenient colorimetric method for detecting Hg²⁺ was developed on the basis of these studies. The Fe₃O₄@Co₃O₄/CN colorimetric sensing process for the detection of mercury ions was as follows: The Fe₃O₄@Co₃O₄/CN concentration was 50 μg·mL⁻¹ and 0.1 mM TMB, and different concentrations of Hg²⁺ standard solutions were added to the 0.1 M sodium acetate buffer solution (pH 3.5). The total volume of the reaction solution was 4 mL, and it was incubated in a water bath at 40 °C for 15 min in the dark.

The water from the Canal River and Hun River comes from Shenyang (Liaoning China), and the water from the Hongqi Canal Lake and Bosong Lake was obtained from Benxi (Liaoning China). Water samples from four different sources were filtered through a 0.22 µm membrane to remove insoluble impurities from the water samples. The water sample was added to the system containing nanozymes, TMB, and a sodium acetate buffer solution, and the reaction was carried out at 40 °C for 15 min. The total volume was 4 mL, and the absorbance value at 652 nm was measured.

3. Results and Discussion

3.1. Synthesis of Fe₃O₄@Co₃O₄/CN

Fe₃O₄@Co₃O₄/CN was obtained via direct calcination pyrolysis under a N₂ atmosphere. By synthesizing Fe₃O₄@Co₃O₄/CN in this manner, the original enzyme activity was maintained, and at the same time, N-crosslinked porous C was formed, which facilitated the process of the catalytic reaction. In order to obtain the best oxidase-like activity, the calcination temperature and time for the calcination process of Fe₃O₄-PVP@ZIF-67 were investigated. The calcination temperature varied from 500 °C to 900 °C, while the calcination time varied from 2 h to 6 h. The temperature and time of calcination mainly affect carbonization. Temperatures that are too low or time periods that are too short will cause insufficient carbonization and insufficient exposure to the active site. Temperatures that are too high and time periods that are too long can cause excessive carbonization, causing the MOF’s skeleton to collapse and affect the catalytic activity of nanozymes. As shown in Figure S1, the strongest Fe₃O₄@Co₃O₄/CN oxidase-like activity was found when the temperature was at 800 °C, and the calcination time was 3 h.

3.2. Characterization of Fe₃O₄@Co₃O₄/CN

The structure and morphology of Fe₃O₄@Co₃O₄/CN were analyzed using various characterization tools. Firstly, the morphology of the nanozyme was observed via scanning electron microscopy (SEM). SEM (Figure 1a,b) showed the morphology of Fe₃O₄-PVP@ZIF-67 before (Figure 1a) and after calcination (Figure 1b). It can be observed that the surface of Fe₃O₄-PVP@ZIF-67 was smooth and relatively homogeneous in size, and the structure of ZIF still maintains its three-dimensional shape. After heat treatment at 800 °C for 3 h under a N₂ atmosphere, the surface of the formed Fe₃O₄@Co₃O₄/CN was rough and uneven in size, and the structure of ZIF collapsed or even disintegrated, forming larger particles. Next, the phase and crystalline shape of the material before and after calcination were analyzed using XRD spectroscopy (Figure 1c). It was observed that the diffraction peaks at 30.2°, 35.6°, 53.6°, and 57.2° correspond to the (220), (311), (422), and (511) crystal planes of Fe₃O₄ (JCPDS PDF No. 19-0629), respectively, and the diffraction peaks at 44.7° and 65° were for Co and Co₃O₄. The presence of cobalt oxide was attributed to the oxidation of the metal Co on the surface in air, and Co²⁺ will be reduced by the carbonized organic linker, thus anchoring the atomically dispersed Co atoms to the nitrogen-doped porous carbon. In the FTIR spectrum (Figure 1d), Fe₃O₄-PVP@ZIF-67 and Fe₃O₄@Co₃O₄/CN both exhibited 588 cm⁻¹, which can be attributed to the Fe-O peak in Fe₃O₄. The 3430 cm⁻¹ band was attributed to the hydroxyl stretching vibration peak of H₂O. The 1630 cm⁻¹ band was ascribed to the C=N stretching vibration due to the structure of 2-MIM of Fe₃O₄-PVP@ZIF-67, while 1110 cm⁻¹ further indicated the existence of C-O-Co stretching vibrations in Fe₃O₄@Co₃O₄/CN. Finally, the saturation magnetization intensity results of Fe₃O₄-PVP@ZIF-67 and Fe₃O₄@Co₃O₄/CN are shown in Figure 1e. They displayed superparamagnetic behavior, and the saturation magnetization values were 68.7 and 95.4 emu·g⁻¹, respectively. The Co on the surface of nanozymes was oxidized by air to form Co₃O₄ with superparamagnetic properties, which increased the magnetic properties of nanozymes. The above characterization results demonstrated the successful synthesis of Fe₃O₄@Co₃O₄/CN.

3.3. Intrinsic Dual Enzyme-like Activities of Fe₃O₄@Co₃O₄/CN

In order to investigate the oxidase activity of Fe₃O₄@Co₃O₄/CN, a series of experiments were conducted using TMB as the chromogenic substrate (Figure 2). As shown in Figure 2, curves 1–5 (Figure 2) had no absorption at 652 nm, while curves 6–7 (Figure 2) produced absorptions of different intensities. In Figure 2, curves 1–5 (Figure 2) illustrated that the reaction systems of Fe₃O₄@Co₃O₄/CN alone, the substrate TMB, and H₂O₂ all did not produce UV absorption. In addition, curves 6–7 (Figure 2) showed that Fe₃O₄@Co₃O₄/CN had both oxidase activity and peroxidase activity, and the oxidase activity was superior to peroxidase activity. Furthermore, as displayed in Figure 2, curves 8–9 (Figure 2) showed that the dual enzyme-like activities of Fe₃O₄-PVP@ZIF-67 were all lower than those of Fe₃O₄@Co₃O₄/CN. The main reason for this result was that Fe₃O₄@Co₃O₄/CN retained its skeletal structure before calcination, which prevents the aggregation of nanoparticles and exposes more active sites of the nanozyme, substantially enhancing the intrinsic dual enzyme-mimetic activities. Based on the oxidase-like activity of Fe₃O₄@Co₃O₄/CN, subsequent experiments were performed to detect Hg²⁺, avoiding the use of the unstable explosion-prone H₂O₂ reagent, which decomposes easily.

Moreover, the oxidase-like activity of Fe₃O₄@Co₃O₄/CN was more dependent on pH, temperatures, reaction times, and nanozyme concentrations. Therefore, the main influencing factors were examined for the above (Figures S2 and S3). The highest oxidase-like activity of Fe₃O₄@Co₃O₄/CN was obtained under the following conditions: TMB (0.1 mM), pH (3.5), incubation temperature (40 °C), incubation time (15 min), and nanozyme concentration (50 μg·mL⁻¹). Compared with Fe₃O₄-PVP@ZIF-67, Fe₃O₄@Co₃O₄/CN not only enhanced dual enzyme-mimetic activities but also shortened the catalytic time, and the related results are shown in Figure S2 and Table S1.

3.4. Steady-State Kinetic Assay of Fe₃O₄@Co₃O₄/CN

To initially explore the catalytic mechanism of nanozymes, kinetic studies of enzymatic behavior were conducted. A typical Michaelis–Menten curve was obtained by varying the TMB concentration under optimal catalytic conditions (Figures S4 and S5). K_m can be used as a measure of the tightness of the enzyme–substrate binding. That is, the smaller the K_m, the smaller the concentration of substrates required to reach half of the maximum reaction rate, indicating a higher affinity of the enzyme relative to the substrate. K_m and V_max were calculated using Lineweaver–Burk plots, and they are listed in Table S2. The higher affinity of Fe₃O₄@Co₃O₄/CN for TMB compared to Fe₃O₄-PVP@ZIF-67 (The K_m value for Fe₃O₄@Co₃O₄/CN (0.28 mM) is approximately 4.6 times lower than the K_m value for Fe₃O₄-PVP@ZIF-67 (1.3 mM)). Fe₃O₄@Co₃O₄/CN had a stronger affinity for TMB and faster reaction rates than most nanozymes, offering a clear advantage as an oxidase mimic.

3.5. Catalytic Mechanism of Fe₃O₄@Co₃O₄/CN

According to the literature, the oxidase-like activity of Fe₃O₄@Co₃O₄/CN may originate from the catalytic capacity of reactive oxygen species (ROS) for TMB [34]. In order to further explore the catalytic mechanism of nanozymes, the ROS species in the system were investigated. P-benzoquinone, NaN₃, and thiourea as trap** agents were used for the capture of superoxide radicals (·O₂⁻), singly linear molecular oxygen atoms (¹O₂), and hydroxyl radicals (·OH), respectively. The results are shown in Figure S6a in Supplementary Materials (SM), Text S7. Compared with the blank, all three different concentrations of scavengers showed some inhibition of oxidase-like activity, indicating that the system produced three types of ROS: ¹O₂, ·OH, and ·O₂⁻. In addition to this, ·O₂⁻-dependent experiments were carried out for the catalytic oxidation of TMB (Figure S6b). The catalytic activity of Fe₃O₄@Co₃O₄/CN was significantly inhibited under N₂-saturated conditions; however, catalytic activities were significantly enhanced under O₂-saturated conditions, which proved that O₂ played a key role in the oxidation of TMB.

3.6. Analytical Performance of Fe₃O₄@Co₃O₄/CN Oxidase Mimic for Hg²⁺ Sensing

3.6.1. Linearity and LOD of GSH and Mercury(II)

GSH would inhibit the oxidase-like activity of the Fe₃O₄@Co₃O₄/CN nanozyme. It can be observed in Figure 3a,b (inset) that within the concentration range of 0–50 μM, with the increase in GSH concentration, the absorbance value of oxTMB at 652 nm became smaller and smaller, and the blue color became lighter and lighter until it was colorless. The concentration of GSH was linear within the range of 1–50 μM. In addition, the limit of detection ($LOD=\frac{3.3\delta }{S}$) for GSH detection via colorimetric sensing was calculated to be 0.966 μM based on (where δ is the standard deviation of 11 blank samples, and S is the slope of the standard curve).

In contrast, with respect to the existence of mercury(II), GSH binds to mercury(II) due to the strong affinity between mercury(II) and -SH and the loss of inhibition; thus, Fe₃O₄@Co₃O₄/CN catalyzes the colorless TMB to become the blue oxTMB. Based on the above principle, a colorimetric sensing method for the determination of the mercury ion was established. As shown in Figure 3c,d(inset), within the concentration range of 0–15 μM, with the increase in mercury(II) concentrations, the absorbance of the GSH-TMB-Fe₃O₄@Co₃O₄/CN system at 652 nm gradually increased, and the color slowly changed from colorless to blue. Meanwhile, the increase in absorbance at 652 nm (ΔA = A − A₀, where A₀ and A are absorbances without and with mercury(II), respectively) increased with an increase in mercury(II) concentrations from 0 to 15 μM. As shown in Figure 3c,d, within the range of 0.1–15 μM, the linear relationship between ΔA and the mercury concentration was good: ΔA = 0.02473C_Hg2+ + 0.04545 (r = 0.9956). The limit of detection ($LOD=\frac{3.3\delta }{S}$) for mercury(II) was 0.017 μM, and the limit of quantitation ($LOQ=\frac{10\delta }{S}$) was 0.052 μM. It is worth mentioning that the detection performance of the colorimetric sensing platform for Hg²⁺ based on Fe₃O₄@Co₃O₄/CN is satisfactory compared to the sensing platforms reported in the literature (

Table 1. Comparison of different colorimetric methods as the sensing platforms for Hg²⁺ detection.

Materials	Test Standard	Linear Range (μM)	LOD (μM)	Reference
Fe3O4@Co3O4/CN	Oxidase-like activity	0.1–15	0.017	This work
AuNPs	Dispersion and surface properties of AuNPs with direct response to Hg2+	1–30	0.3	[11]
CS-SeNPs	Oxidase-like activity	0.1–2.5	0.12	[26]
Hollow MnFeO oxide	Oxidase-like activity	0.1–15	0.02	[32]
Ag3PO4 microcubes	Oxidase-like activity	0.1–7.0	0.02	[35]
Ag@Ag2WO4	Oxidase-like activity	0.25–8.0	0.0016	[36]
AgNPs	Oxidase-mimic activity	0–600	0.03	[37]
AgNPs	Redox reaction	0–120	7.47	[38]

).

3.6.2. Selectivity of the Proposed Method

The strong immunity relative to interference determines the reliability of the method in the detection of real samples. The common ions in water (K⁺, Ca²⁺, Co²⁺, NH₄⁺, Ni⁺, Ba²⁺, Zn²⁺, Na⁺, Al³⁺, Mg²⁺, Cu²⁺, Mn²⁺, Fe³⁺, and La²⁺) were selected as interfering substances for anti-interference experiments. As shown in Figure 4, the interfering ion alone has no significant absorption at 652 nm even if their concentration was at the 100 μM level, 10 times higher than the concentration of Hg²⁺. On the contrary, in the simultaneous presence of interfering ions and Hg²⁺, there was significant absorption at 652 nm, and the interferents almost did not affect the detection of Hg²⁺. It can be observed that the established colorimetric sensing method for the detection of Hg²⁺ had a strong selectivity.

3.6.3. Accuracy and Practicability

A standard addition method was used to verify the accuracy of the colorimetric sensing method for the detection of Hg²⁺. The Hg²⁺ concentrations were calculated from the accompanying standard curves and the spiked recoveries, and RSD was obtained. The results are shown in Table S4. The mean recovery of the nine samples was 96.3%, with an RSD of 3.1%. It is demonstrated that the proposed colorimetric sensing method has good accuracy and precision for the detection of Hg²⁺. To explore the feasibility of the method to Hg²⁺ in real water samples, four water samples from the local river lake and laboratory tap water were collected and detected using the proposed method. The results are listed in Table S5. No Hg²⁺ was detected in all water samples.

3.6.4. Reusability of Fe₃O₄@Co₃O₄/CN

Fe₃O₄@Co₃O₄/CN can be easily recovered due to its outstanding superparamagnetism. The reusability of Fe₃O₄@Co₃O₄/CN was studied according to the sample determination process. The material was recovered using magnets after each experiment. A total of 10 experiments were performed. The results are shown in Figure 5. It can be observed that there was almost no difference in the absorbance values obtained from each cycle. This showed that the activity had little change after repeated use of the material, which fully indicated that the reusability of Fe₃O₄@Co₃O₄/CN was extremely good. This feature is very beneficial for the development of an economical commercial testing platform.

3.6.5. Stability of Fe₃O₄@Co₃O₄/CN

As a nanozyme, Fe₃O₄@Co₃O₄/CN was expected to be more stable than natural enzymes. Thus, the stability of the nanozyme was further investigated by exposing the material to different temperatures (20~90 °C) and different pH (2.5~10.5) levels for 1 h. After the materials were recovered, the absorbance was measured under optimal conditions of oxidase-like activity. As shown in Figure 6a, the absorbance was slightly reduced in acidic environments with a pH of <5, and it was almost unchanged within the range of pH5~10.5, indicating that the material has good stability within a wide pH range. From Figure 6b, it can be observed that the activity of the nanozyme had no evident changes in temperature ranges of less than 70 °C, and it still exhibited high activity at high temperatures of 80 °C and 90 °C. This shows that the material can withstand a certain high temperature. Furthermore, the storage stability of the material was also investigated, and the results are shown in Figure 6c,d. There was almost no change in nanozyme activity after incubation in pure water for 30 days and storage at room temperature for 7 months. The above results show that Fe₃O₄@Co₃O₄/CN had excellent stability, which lays a foundation for its wide application.

4. Conclusions

Fe₃O₄@Co₃O₄/CN, with remarkable dual enzyme-like activities and superparamagnetism, was successfully synthesized using a high-temperature carbonization strategy. In addition, Fe₃O₄@Co₃O₄/CN followed typical Michaelis–Menten kinetics with improved stability and reusability. Furthermore, the catalytic mechanism of the nanozyme was shown to involve three reactive oxygen species (¹O₂, ·OH, and ·O₂⁻). Then, a simple, sensitive, and selective colorimetric biosensor for mercury(II) was developed based on the remarkable oxidase-like activity of Fe₃O₄@Co₃O₄/CN. Our work provided a feasible synthesis method for the preparation of efficient and stable nanozymes with potential applications in the detection and monitoring of mercury ions in real water samples.