Oxidase-Like Catalytic Performance of Nano-MnO2 and Its Potential Application for Metal Ions Detection in Water

Certain nano-scale metal oxides exhibiting the intrinsic enzyme-like reactivity had been used for environment monitoring. Herein, we evaluated the oxidase-mimicking activity of environmentally relevant nano-MnO2 and its sensitivity to the presence of metal ions, and particularly, the use of MnO2 nanozyme to potentially detect Cu2+, Zn2+, Mn2+, and Fe2+ in water. The results indicated the oxidase-like activity of nano-MnO2 at acidic pH-driven oxidation of 2,6-dimethoxyphenol (2,6-DMP) via a single-electron transfer process, leading to the formation of a yellow product. Notably, the presence of Cu2+ and Mn2+ heightened the oxidase-mimicking activity of nano-MnO2 at 25°C and pH 3.8, showing that Cu2+ and Mn2+ could modify the reactive sites of nano-MnO2 surface to ameliorate its catalytic activity, while the activity of MnO2 nanozyme in systems with Zn2+ and Fe2+ was impeded probably because of the strong affinity of Zn2+ and Fe2+ toward nano-MnO2 surface. Based on these effects, we designed a procedure to use MnO2 nanozyme to, respectively, detect Cu2+, Zn2+, Mn2+, and Fe2+ in the real water samples. MnO2 nanozyme-based detecting systems achieved high accuracy (relative errors: 2.2–26.1%) and recovery (93.0–124.0%) for detection of the four metal ions, respectively. Such cost-effective detecting systems may provide a potential application for quantitative determination of metal ions in real water environmental samples.


Introduction
In recent years, considerable attention has been paid to the applications of artificial nanomaterials as nanozymes in mimicking the intrinsic catalytic function of natural enzymes due to their unique structural, electrical, and optical properties, as well as remarkable catalytic activities [1][2][3]. Compared with the natural enzymes, the artificial nanozymes exhibited higher robustness and stability under harsh conditions, lower production cost, simpler storage conditions, and more effective catalytic activity [4][5][6]. At present, nanozymes are primarily composed of artificial metal and metal oxide nanomaterials that can mimic the catalytic activities of natural peroxidases and/or oxidases [3,7]. For instance, the intrinsic peroxidase-/oxidase-like activities of Au-Ag, CeO 2 , MnFe 2 O 4 , NiO, and V 2 O 5 nanoparticles have been used in various applications ranging from biosensing and immunoassay to environment monitoring [3,[7][8][9][10]. Liu et al. reported the oxidase-mimicking activity of CeO 2 nanoparticles by fluoride capping, such nanozymes could detect micromolar levels of F − in water and toothpastes [11].
It is well documented that MnO 2 nanomaterial had the intrinsic enzyme-like activity to catalyze the chromogenic reaction of substrates, which could be used as a nanozyme indicator for bioimaging, biosensing, and delivery of singlestranded DNA and drugs [3,12,13]. In particular, chromogenic reactions by nano-MnO 2 have been developed using dissolved O 2 as the oxidant, avoiding the use of H 2 O 2 [14], thus providing easy and rapid detecting systems for quantitative analysis of any substances that can serve either as the accelerator or inhibitor of the chromogenic reactions [15,16]. Such systems could be used for real environmental water samples, but their potential application for quantitative determination of metal ions has been rarely explored. e contamination of metal ions has always been a focus of concern [17]. Toxic metal ions (such as Cu 2+ , Zn 2+ , Pb 2+ , and Fe 2+ ) having toxicity level greater than safety levels can cause acute toxicities to most aquatic biota [18]. us, having ways that can easily and rapidly detect these metal ions in water matrices is vital to protect wild species and human health. A instrumental method can be used to directly detect these metal ions in water samples, such as inductively coupled plasma mass spectrometry (ICPMS), but such methods are usually expensive and time-consuming and require expertise to operate [19,20].
Recently, the enzyme-like activity of environmentally relevant nano-MnO 2 has proven to be highly effective for sensing applications [16,21,22]. In this study, nano-MnO 2 was chosen as the natural oxidase mimic owing to its outstanding redox chemistry, stability, and biocompatibility properties [23][24][25]. We systematically evaluated the oxidaselike activity of nano-MnO 2 in catalyzing the chromogenic reaction of 2,6-dimethoxyphenol (2,6-DMP), and identified the influence of Cu 2+ , Zn 2+ , Mn 2+ , and Fe 2+ , based on which methods were developed to, respectively, detect these metal ions in environmental water samples using MnO 2 nanozyme-2,6-DMP detecting systems.

Chemicals and Materials.
Nano-scale MnO 2 (≥99.9%) was obtained from DK Nano Technology Co., Ltd. (Beijing, China). e characteristics of nano-MnO 2 are shown in Figure 1. e size and morphology of the nano-MnO 2 were analyzed using a transmission electron microscope (TEM, JEM-200CX). e spectral characteristics of nano-MnO 2 were investigated using a UV-Vis spectroscope (Shimadzu, UV-2550) and a Fourier-transform infrared spectroscope ( ermo Scientific, NICOLET iS50 FTIR). e phase of the nano-MnO 2 was measured over the 2θ range from 5 to 85 degrees using an X-ray diffractometer (XRD, ermo X'TRA).
e buffer used in this study was a citratephosphate buffer solution (C-PBS: 10 mmol·L − 1 citric acid and 10 mmol·L − 1 Na 2 HPO 4 , pH 3.8) adjusted with HCl and NaOH. All the other chemicals were of analytical reagent grade and used as received.

Assessment of the Enzyme-Like Activity of Nano-MnO 2 .
To assess the enzyme-like activity of nano-MnO 2 , MnO 2 nanoparticles were tested in 10 mL of a C-PBS (10 mmol·L − 1 , pH 3.8) buffer at room temperature (25°C) containing 1.0 mmol·L − 1 2,6-DMP as the chromogenic substrate and naturally dissolved O 2 as the cofactor [14,26]. After the nano-MnO 2 (0.1 mg·mL − 1 ) had been mixed thoroughly with the 2,6-DMP reaction solution, the absorbance was immediately measured at 468 nm using a UV-Vis spectrophotometer (Shanghai Lengguang 722S) in a quartz cuvette with a 1 cm light path. e solution was monitored every 20 s for 3 min by recording the change of absorbance value at 468 nm. One unit of nano-MnO 2 activity (U·mL − 1 ) is defined as the amount of nanozyme that causes one unit of absorbance change per minute at 468 nm in C-PBS (10 mmol·L − 1 , pH 3.8) buffer containing 1.0 mmol·L − 1 2,6-DMP. erefore, the oxidase-mimicking activity of nano-MnO 2 can be calculated through the rate of absorbance change. e same solution free of nano-MnO 2 was used as the blank control. All experiments were performed in triplicate.

Effect of Different Factors on the Enzyme-Like Activity of Nano-MnO 2 .
To evaluate the influence of nano-MnO 2 dosage on 2,6-DMP oxidation, the reaction was conducted in a 50 mL flask containing 1.0 mmol·L − 1 2,6-DMP and nano-MnO 2 varying between 0.005 and 0.32 mg·mL − 1 in 10 mL C-PBS (10 mmol·L − 1 ) at 25°C and pH 3.8. e effect of the substrate concentration on the chromogenic reaction was also performed in a 50 mL flask containing 0.1 mg·mL − 1 nano-MnO 2 and 2,6-DMP at a concentration varying between 0.005 and 1.0 mmol·L − 1 in 10 mL C-PBS. e reaction kinetics parameters K m and v max were calculated by the Lineweaver-Burk plot of the Michaelis-Menten kinetics equation: where v is the reaction velocity, [S] is the substrate concentration, K m is the Michaelis constant, and v max is the maximal reaction velocity. Experimental procedures similar to those described above were used to explore the effects of pH and temperature on the enzyme-like activity of nano-MnO 2 . e reactions were carried at different pH and a wide range of temperature. For studying the pH effect, 10 mL of 10 mmol·L − 1 C-PBS (pH 2.0-10.0) buffer containing 1.0 mmol·L − 1 2,6-DMP was mixed with 0.1 mg·mL − 1 nano-MnO 2 at room temperature (25°C). For studying the effect of temperature, 10 mL of 10 mmol·L − 1 C-PBS (pH 3.8) buffer containing 1.0 mmol·L − 1 2,6-DMP was mixed with 0.1 mg·mL − 1 nano-MnO 2 at a temperature ranging from 10°C to 90°C. Absorbance was recorded at 468 nm at 20 s intervals. All experiments were performed in triplicate.

Oxidase-Like Activity of Nano-MnO 2 .
To assess the intrinsic enzyme-mimicking activity of nano-MnO 2 , 2,6-DMP was chosen as the chromogenic substrate in the standard oxidation reaction, and the reaction kinetics was tested at 468 nm corresponding to the oxidized 2,6-DMP. e change of absorbance over time by the oxidation of 2,6-DMP in C-PBS buffer at 25°C and pH 3.8 is shown in Figure 2. Nano-MnO 2 could catalyze the colorless 2,6-DMP to form a chromogenic product (a yellow product, i.e., 3,3′,5,5′-tetramethyl-4,4′-diphenoquinone) with a change in absorbance via the radical-based C-C self-coupling mechanism, like laccase-mediated oxidative coupling reactions of 2,6-DMP under the same conditions [26,27]. e absorbance changes linearly with time under the tested conditions (R 2 > 0.99), and the oxidase-like activity of nano-MnO 2 was calculated to be 0.047 U·mL − 1 . e oxidative coupling of 2,6-DMP catalyzed by nano-MnO 2 was described as follows: first, 2,6-DMP was adsorbed onto the reactive sites of nano-MnO 2 surface, followed by the single-electron oxidation of 2,6-DMP by nano-MnO 2 , leading to the formation of chromogenic product and the release of Mn 2+ from the nanoparticle surface [14,28]. e role of dissolved O 2 in the oxidation of 2,6-DMP was evaluated by purging the reaction solution with N 2 , resulting in a decrease on the oxidase-like activity of nano-MnO 2 . is revealed that dissolved O 2 acted as an electron acceptor in the catalytic reactions [14,29]. is result is in agreement with an earlier report that indicated the oxidation of a substrate in the absence of H 2 O 2 via bovine serum albumin-(BSA-) stabilized MnO 2 nanoparticles [30]. Additionally, the stability of nano-MnO 2 in the reaction system was also studied over a one-month storage period. With the increase in storage time, the release of Mn 2+ increased mildly, but no significant difference in the oxidation of 2,6-DMP was detected, implying that the capacity of nano-MnO 2 to oxidize 2,6-DMP exhibits a high stability. ese results demonstrated that nano-MnO 2 possessed a stable oxidase-like activity to catalyze the chromogenic reaction of 2,6-DMP at 25°C and pH 3.8 in the absence of H 2 O 2 .

Effects of Nano-MnO 2 and Substrate Concentration on 2,6-DMP Oxidation.
We further assessed the influence of nano-MnO 2 concentration on 2,6-DMP oxidation catalyzed by MnO 2 nanozyme by UV-Vis spectrophotometry. As shown in Figure 3, the oxidation of 2,6-DMP catalyzed by MnO 2 nanozyme showed a distinct absorbance peak at the wavelength of 468 nm, and the increase of this absorbance over time was obvious resulting from 2,6-DMP oxidation ( Figure 2). e variation of the absorbance peak was observed by adding different concentrations of MnO 2 nanozyme (Figure 3). Increasing the concentration of nano-MnO 2 from 0.005 to 0.3 mg·mL − 1 resulted in a liner increase in the oxidase-like activity of nano-MnO 2 (0.002-0.126 U·mL − 1 ) in oxidizing 2,6-DMP ( Figure 4). According to the correlation of the nano-MnO 2 concentration and its oxidase-like activity, the apparent pseudosecond-order rate constant was determined to be 0.445 ese results demonstrated that increasing the concentration of nano-MnO 2 facilitated the oxidase-like activity of nano-MnO 2 to catalyze the oxidation of 2,6-DMP.
For discussing the catalytic mechanism and obtaining the steady-state kinetic parameters, the initial reaction rate (1 min) of 2,6-DMP oxidation catalyzed by nano-MnO 2 was investigated with the initial 2,6-DMP concentration varying between 0.005 and 0.2 mmol·L − 1 . A hyperbolic relationship between the substrate concentration and the rate of reaction (v) was revealed in Figure 5(a), like the typical Michaelis-Menten curve. e apparent enzyme kinetic parameters such as K m and v max values could be calculated by Lineweaver-Burk plot ( Figure 5(b)). From the kinetic analysis, it was found that MnO 2 nanozyme showed a high affinity towards 2,6-DMP.
e K m and v max values were 0.005 and 0.155 (R 2 � 0.999), respectively. Combining with previous studies on artificial metal oxide-based nanozymes [24,31,32], MnO 2 nanoparticles are promising nanomimetics for oxidase. It is noted that the oxidase-like activity of nano-MnO 2 and the steady-state kinetic parameter values were investigated at an acidic pH (pH 3.8) because of its limited oxidase-like activity at physiological or basic pH.  International Journal of Analytical Chemistry

Effects of pH and Temperature on 2,6-DMP Oxidation.
Similar to the natural oxidase, the catalytic activity of MnO 2 nanozyme is also dependent on pH and temperature. As shown in Figure 6, the catalytic activity of MnO 2 nanozyme decreased with the rise of reaction pH from 2.0 to 7.0, whereas the oxidase-like activity of MnO 2 nanozyme increased with the rise of reaction temperature from 10°C to 90°C. It was found that only 0.022 U·mL − 1 of nano-MnO 2 activity was retained at pH 7.0, while 0.205 U·mL − 1 of activity was retained even at 90°C. As the reaction pH increasing from 7.0 to 10.0, the oxidase-like activity of nano-MnO 2 had not exhibited an obvious variation. As the temperature increased from 10°C to 25°C, the catalytic activity of nano-MnO 2 was mildly enhanced. It was noted that as the temperature increased from 30°C to 90°C, the catalytic activity rapidly increased. Temperature varying in the range of 10-25°C had little impact on the final colorimetric signal. Change in pH and temperature had not resulted in inactivation of MnO 2 nanozyme. ese results indicated that the oxidase-like activity of nano-MnO 2 exhibited a wide range of pH and thermal stability, unlike the natural oxidase [33,34].

Metal Ions Induced the Effect of MnO 2 Nanozyme Activity.
Simply and accurately detecting metal ions is of great significance in the aqueous environment. Several nanozymes had been used to detect metal ions (i.e., Hg 2+ and Pb 2+ ) due to their intrinsic advantages and high stability under harsh conditions [35][36][37]. In this study, the selectivity of MnO 2 nanozyme activity was evaluated in the presence of various metal ions including Mg 2+ , Cu 2+ , Al 3+ , Zn 2+ , Mn 2+ , Fe 2+ , and Pb 2+ in 10 mL C-PBS buffer at 25°C and pH 3.8. As shown in Figure 7, the oxidase-like activity of nano-MnO 2 was 0. Previous studies had also indicated that certain metal ions could effectively upregulate/downregulate the activity of nanozymes through surface deposition and metallophilic interactions [38][39][40]. For MnO 2 nanozyme detecting systems, the substrate (2,6-DMP) was transformed into a chromogenic product, serving as a signal amplifier. e presence of Cu 2+ and Mn 2+ enhanced the activity of MnO 2 nanozyme, likely because these metal ions modified the reactive sites of nano-MnO 2 surface [41,42]. First, Cu 2+ and/ or Mn 2+ ions reacted with citrate to form metal ion-citrate complex, subsequently the complex dispersed onto the surface of nano-MnO 2 , and thus changed the surface properties of nano-MnO 2 , thereby enhancing its oxidaselike activity [43,44]. On the contrary, the suppressive activity on MnO 2 nanozyme in the presence of Zn 2+ and Fe 2+ occurred probably owing to the strong affinity of Zn 2+ and Fe 2+ toward the nano-MnO 2 surface via the electrostatic attractions or metal ion-multivalent Mn interactions [36,39,40]. e binding affinity of MnO 2 nanozyme for Zn 2+ and Fe 2+ was very high. e adsorption of Zn 2+ and Fe 2+ onto the MnO 2 nanozyme impeded the electron transfer to 2,6-DMP, thus diminishing the oxidase-like activity of nano-MnO 2 [39]. Additionally, the control samples free of MnO 2 nanozyme with the metal ion present did not show the oxidase-like activity towards O 2 -2,6-DMP during the incubation period.

MnO 2 Nanozyme-Based Reaction Systems for Detecting
Cu 2+ , Zn 2+ , Mn 2+ , or Fe 2+ . As shown in Figure 8, MnO 2 nanozyme-sensing systems were carried out by, respectively,  To further investigate the possible interaction mechanism between metal ions and MnO 2 nanozyme, a differential UV-Vis spectrometry approach was performed [45]. e differential absorbance spectrum (DAS) could be calculated by the following equation: where A mixture , A 2,6-DMP , and A metal ion are, respectively, the absorbance at 250-600 nm wavelength of the mixture solution, and the corresponding reference 2,6-DMP and metal ion solution. As shown in Figure 9, the DAS of four reaction systems had an intensive negative peak at 272 nm and two intensive positive peaks, respectively, at 320 and 468 nm, implying that the change of electronic density in the molecules caused by the formation of a complex and/or metal ion-multivalent Mn interactions in C-PBS buffer. On the one hand, the formation of complex between Cu 2+ /Mn 2+ and citrate changed the surface properties of MnO 2 nanozyme, thus facilitating its oxidase-like activity [43,44,46]. On the other hand, Zn 2+ and Fe 2+ were bound to the reactive sites of MnO 2 nanozyme surface, leading to the hindrance of electron transfer between the MnO 2 nanozyme and 2,6-DMP, consequently restraining the activity of MnO 2 nanozyme [39,40].

Detection of Metal Ions in Real Water
Samples. In order to verify the metal sensing ability of MnO 2 nanozyme for real environmental water samples, tests were performed with different concentrations of Cu 2+ , Zn 2+ , Mn 2+ , or Fe 2+ spiked to pond water samples 1 and 2 from Anhui Agricultural University. First, samples were diluted 10-fold with C-PBS buffer (pH 3.8) to minimize the matrix effect. Subsequently, Cu 2+ , Zn 2+ , Mn 2+ , or Fe 2+ at a concentration of 0.002-0.25 mmol·L − 1 were spiked to the pond water samples. As shown in Table 1, the recoveries were 93.0-124.0% for 0.002-0.25 mmol·L − 1 metal ions (i.e., Cu 2+ , Zn 2+ , Mn 2+ , and Fe 2+ ) that were spiked to the pond water 1 and 2. e concentrations of Cu 2+ , Zn 2+ , Mn 2+ , and Fe 2+ in the pond water samples 1 and 2 were also determined by ICPMS, which did not show significant difference from that obtained by the MnO 2 nanozyme-detecting systems. In addition, the nanozyme-sensing method exhibited stable performance at a broad range of pH and temperature, convenient for experimental applications. It is noteworthy that the response of the MnO 2 nanozyme-sensing systems to Zn 2+ and Fe 2+ at high concentrations can be directly observed with the naked eye.
ese results confirmed that the MnO 2 nanozyme-2,6-DMP-sensing systems may be applicable to real water environmental samples for easily and rapidly quantifying Cu 2+ , Zn 2+ , Mn 2+ , or Fe 2+ . Even so, how to improve the selectivity of MnO 2 nanozyme for metal ions detection in water is still crucial. To achieve that, two of the following main issues need to be resolved. One is studying the catalytic performance and steady-state kinetics to uncover the interaction mechanism between MnO 2 nanozyme and metal ions, and the other is modifying the surface of MnO 2 nanozyme to improve its catalytic activity and environmental application in real water [3,13,44,47,48].

Conclusions
In this study, nano-MnO 2 was used as an oxidase mimetic to catalyze the chromogenic reaction of 2,6-DMP in C-PBS buffer.
e results indicated that nano-MnO 2 possessed the oxidaselike activity with the K m and v max values of 0.005 and 0.155 (R 2 � 0.999), respectively, at 25°C and pH 3.8. Additionally, the effect of metal ions on this colorimetric reaction catalyzed by MnO 2 nanozyme was explored, based on which it was found that this reaction system could be used to, respectively, detect Cu 2+ , Zn 2+ , Mn 2+ , and Fe 2+ in aqueous solution without significant interference from other factors. e detection limit for the four metal ions was less than 0.002 mmol·L − 1 and the linear response range was 0.002-0.25 mmol·L − 1 . Use of this detecting system was demonstrated with real environmental water samples, and the results indicated that the MnO 2 nanozymebased sensing was simple and rapid for quantitative determination of Cu 2+ , Zn 2+ , Mn 2+ , and Fe 2+ . It is noted that MnO 2 nanozyme was unable to determine ultralow metal ion concentration; thus, a more sensitive detecting assay should be developed in the follow-up study.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no competing financial interests.