Peroxidase-Like Metal-Based Nanozymes: Synthesis, Catalytic Properties, and Analytical Application

: Nanozymes (NZs) are nanostructured artiﬁcial enzymes that mimic catalytic properties of natural enzymes. The NZs have essential advantages over natural enzymes, namely low preparation costs, stability, high surface area, self-assembling capability, size and composition-dependent activities, broad possibility for modiﬁcation, and biocompatibility. NZs have wide potential practical applications as catalysts in biosensorics, fuel-cell technology, environmental biotechnology, and medicine. Most known NZs are mimetics of oxidoreductases or hydrolases. The present work aimed to obtain effective artiﬁcial peroxidase (PO)-like NZs (nanoPOs), to characterize them, and to estimate the prospects of their analytical application. NanoPOs were synthesized using a number of nanoparticles (NPs) of transition and noble metals and were screened for their catalytic activity in solution and on electrodes. The most effective nanoPOs were chosen as NZs and characterized by their catalytic activity. Kinetic parameters, size, and structure of the best nanoPOs (Cu/Ce S ) were determined. Cu/Ce S -based sensor for H 2 O 2 determination showed high sensitivity (1890 A · M − 1 · m − 2 ) and broad linear range (1.5–20,000 µ M). The possibility to apply Cu/Ce S -NZ as a selective layer in an amperometric sensor for hydrogen-peroxide analysis of commercial disinfectant samples was demonstrated.

PO is widely used in different fields of science and industry, especially in analytics for H 2 O 2 determination [1]. Many natural enzymes (oxidases) produce H 2 O 2 as a byproduct of their enzymatic reactions, so that detection of the target substrates can be performed by The aim of this work was to obtain effective NZs having PO-like activity, to characterize these nanoperoxidase NZs (nanoPOs), and to estimate the prospects of their analytical application.
The novelty of the current research is a synthesis of a broad row of hybrid metal nanoparticles (NPs) containing transition and noble metals, and a comparative study of their catalytic activity when immobilized on electrodes as well as in a solution. The latter issue is almost not related to in literature. Kinetic parameters, size, and structure of the most effective nanoPOs were determined. The Cu/Ce-based sensor for H 2 O 2 determination showed the highest sensitivity (1890 A·M −1 ·m −2 ), a broad linear range (1.5-20,000 µM), and a high current response (786 µA) upon H 2 O 2 addition. The possibility to apply Cu/Ce S -NZ as a selective layer in an amperometric sensor for hydrogen peroxide analysis in commercial disinfectant samples was demonstrated.

Synthesis of NPs
NPs were synthesized by the reduction of metal ions from appropriate salts, according to the methods used in our modifications [41][42][43][44]. The conditions of NP synthesis are presented in Table 1. NPs were collected by centrifugation under 10,000× g for 40 min (Hettich Micro-22R centrifuge), washed twice with water, and precipitated by centrifugation. Pellets were suspended in 0.2 mL of water and stored until use at +4 • C.

Morphological Analysis of NPs Using Scanning Electron Microscopy (SEM)
Morphological analyses of the samples were performed using a SEM microanalyzer (REMMA-102-02, Sumy, Ukraine). The samples in different dilutions (2 µL) were dropped onto the surface of a silicon wafer and were dried at room temperature. The distance from the last lens of the microscope to the sample (WD) ranged from 17.1 mM to 21.7 mM. The accelerator voltage was in the range of 20 to 40 eV.

Determination of Peroxidase-Like Activity of NPs in Solution
PO-like activity of the NPs was measured by the colorimetric method, with o-dianisidine as a chromogenic substrate in the presence of H 2 O 2. The generated color was determined at 525 nm using a Shimadzu UV1650 PC spectrophotometer (Kyoto, Japan). One unit (U) of PO-like activity was defined as the amount of NZ releasing 1 µMoL H 2 O 2 per 1 min at 30 • C under standard assay conditions.
The procedure of the PO-like activity assay: 10 µL of the aqueous suspension of NPs (1 mg/mL) was incubated in a glass tube with 1 mL of 0.17 mM o-dianisidine in 50 mM NaOAc buffer, pH 4.5 (as a control); and with the same substrate in the presence of 8.8 mM H 2 O 2 (as a substrate for PO). Addition of NPs to the substrate stimulated the development of an orange color over time, indicating an enzymatic reaction. The enzymemimetic activity could be assessed qualitatively with the naked eye and was measured quantitatively with a spectrophotometer. After incubation for an exact time (1-10 min) at 30 • C, and upon appearance of the orange color, the reaction was stopped by the addition of 0.26 mL 12 M HCl. The millimolar extinction coefficient of the resulting pink dye in the acidic solution was 13.38 mM −1 ·cm −1 .

Apparatus, Measurements, and Statistical Analysis
The amperometric sensors were evaluated using constant-potential amperometry in a three-electrode configuration with an Ag/AgCl/KCl (3 M) reference electrode, a Ptwire counter electrode, and a graphite working electrode. Graphite rods (type RW001, 3.05 mM diameter) from Ringsdorff Werke (Bonn, Germany) were sealed in glass tubes using epoxy glue to form disk electrodes. Before sensor preparation, the graphite electrode (GE) was polished with emery paper and a polishing cloth using decreasing particle sizes of alumina paste (Leco, Germany). The polished electrodes were rinsed with water in an ultrasonic bath.
Amperometric measurements were carried out using a CHI 1200A potentiostat (IJ Cambria Scientific, Burry Port, UK) connected to a personal computer and performed in a batch mode under continuous stirring in an electrochemical cell with a 20 mL volume at 25 • C. All the experiments were carried out in triplicate trials. Analytical characteristics of the electrodes were statistically processed using OriginPro 8.5 software. The error bars represent the standard error derived from three independent measurements. Calculation of the apparent Michaelis-Menten constants (K M app ) was performed automatically by this program according to the Lineweaver-Burk equation.

Immobilization of Metallic NPs onto Electrodes, Testing Their Electro-and PO-Like Activity
For construction of the NZ-based electrode, 5 µL of NP suspension (1 mg/mL) was dropped onto the surfaces of GEs. After drying for 10 min at room temperature, the layer of NPs on the electrodes was covered with 5 µL Nafion membrane. The electrodes were washed with corresponding buffer solutions before and after each measurement.
The electrochemical properties of the synthesized NPs were studied by cyclic voltammetry (CV) on the GEs in the range from −1000 to +1000 mV with the scan rate of 50 mV min −1 ; the profiles of amperometric signals in increasing concentrations of H 2 O 2 were compared. The most electroactive NPs, which had the highest PO-like properties, were chosen for further investigation.  (Table 2). These results are in agreement with the data of other researchers [9,10]. The most catalytically active NZs were characterized by SEM coupled with X-ray microanalysis (SEM-XRM). SEM provided information on the size, distribution, and shape of the tested sample. Figure S1 presents the overall morphology of the formed hybrid particles. The XRM images of the synthesized NZs showed the characteristic peaks for metals of the composites.

Obtaining and Characterizing the Best Peroxidase-Like Nanozymes
The SEM imaging and XRM data proved the formation of needlelike hybrid Fe/Ce and Au/Cu micro particles with the size ranging from 10 to 20 µM ( Figure S1). The XRM image of Au/Cu NPs shows the Kα peaks of Cu 0 and Au 0 , confirming the presence of both CuNPs and AuNPs. It was shown that the Fe/Ce and Au/Cu NPs tested look like irregular shaped clusters of microfibrillar structures. The best of catalytically active NZs, namely, Cu/CeS, Au/Cu, Ag/Ce, Fe/Ce, Ag/Zn, Pd/Ce, and Pt/Cu NPs were chosen for further investigation.

Development and Characterization of the NZs-Modified Electrodes
Our next task was to select the most PO-active NPs immobilized on the electrode surface. It is known that modification of an electrode with some metallic NPs can improve the efficiency of an electron transfer, due to an increase in the electrochemically accessible electrode surface area. Numerous NZs/GEs were screened for their ability to decompose hydrogen peroxide. The electrocatalytic activities of the synthesized NZs, while immobilized on the surface of GEs, were tested by CV and chronoamperometry, as described in Section 2.5.2 ( Figure 1 and Figure S2). In our research, the amperometric responses of different NZs/GEs to added portions of H2O2 were compared under mild conditions at the working potential of −50 mV. This rather low potential was chosen from the CV profiles (Figure 1), since the main drawback of many H2O2-sensitive NZ-based electrochemical sensors is their nonselectivity under high positive (or negative) working potentials. H2O2 is able to direct the auto-oxidation on electroactive surfaces at a working potential above +0.4 V, or auto-reduction at −0.4 V or less vs. Ag/AgCl. Moreover, the real samples containing organic compounds may be easily co-oxidized/co-reduced at extreme potentials, which may result in overestimation of the target analytes in the presence of oxygen. To avoid these problems during sensor anal- of many H 2 O 2 -sensitive NZ-based electrochemical sensors is their nonselectivity under high positive (or negative) working potentials. H 2 O 2 is able to direct the auto-oxidation on electroactive surfaces at a working potential above +0.4 V, or auto-reduction at −0.4 V or less vs. Ag/AgCl. Moreover, the real samples containing organic compounds may be easily co-oxidized/co-reduced at extreme potentials, which may result in overestimation of the target analytes in the presence of oxygen. To avoid these problems during sensor analysis, it was important to screen new NZs while working at operating potentials close to zero (0) V vs. Ag/AgCl [12,[43][44][45]. This requirement is especially relevant for the construction of biosensors and their exploitation for analysis of real samples (food products, biological liquids, etc.). Our previous experiments with natural PO as a biorecognition element of amperometric biosensors were also carried out at −50 mV [41][42][43][44].
Using the chronoamperograms, calibration curves were plotted for H 2 O 2 determination by the developed electrodes ( Figure S2). The analytical characteristics of the modified GEs, as deduced from the graphs in comparison with natural PO [42][43][44], are summarized in Table 3. The linear ranges, limits of detection (LOD), and sensitivities of the electrodes modified with nanoPOs were calculated. The limit of blank values (LOB) for NZ was calculated as 0.3 × LOD. The sensitivity of each NZ-modified electrode was measured for an electrode area of 7.30 mM 2 . Table 3. Analytical characteristics of the most effective NZs as PO-mimetic placed on electrode.

Sensitive Film
No. in Table 2 Figure 2 summarizes the data concerning the PO-like properties of the selected NZs, namely, Cu/Ce S , Au/Cu, Ag/Ce, Fe/Ce, Ag/Zn, Pd/Ce, and Pt/Cu. The results in Table 3 demonstrate that Cu/Ce S /GE exhibited the highest sensitivity (1890 A·M −1 ·m −2 ) and current response (786 µA), the lowest LOD (0.42 µM), and a broad linear range (1.5-20,000 µM) upon H 2 O 2 addition. This structure was therefore studied in more detail as the most effective artificial PO immobilized on the electrode. Using the chronoamperograms, calibration curves were plotted for H2O2 determination by the developed electrodes ( Figure S2). The analytical characteristics of the modified GEs, as deduced from the graphs in comparison with natural PO [42][43][44], are summarized in Table 3. The linear ranges, limits of detection (LOD), and sensitivities of the electrodes modified with nanoPOs were calculated. The limit of blank values (LOB) for NZ was calculated as 0.3 × LOD. The sensitivity of each NZ-modified electrode was measured for an electrode area of 7.30 mm 2 . Figure 2 summarizes the data concerning the PO-like properties of the selected NZs, namely, Cu/Ce S , Au/Cu, Ag/Ce, Fe/Ce, Ag/Zn, Pd/Ce, and Pt/Cu. The results in Table 3 demonstrate that Cu/Ce S /GE exhibited the highest sensitivity (1890 A·M⁻ 1 ·m⁻ 2 ) and current response (786 µA), the lowest LOD (0.42 µM), and a broad linear range (1.5-20,000 µM) upon H2O2 addition. This structure was therefore studied in more detail as the most effective artificial PO immobilized on the electrode.

Characterization of the Most Effective Cu/Ce S -Modified Electrode
The Cu/Ce S -NZ with the highest PO-like activity was studied in detail. The Cu/Ce S /GE demonstrated the highest PO-like activity, compared with other NZs and with natural PO ( Figure S2, Tables 3 and 4). The highest current response (I max ) on the tested analyte at substrate saturation of the Cu/Ce S /GE was 157-fold higher, and its sensitivity was 5.4-fold higher, than that of the PO/GE (Table 3). It is worth mentioning that Cu/Ce S has PO-like ability but lacks oxidase (laccase)-like properties. This conclusion was reached from the results of colorimetric tests using o-dianisidine in the presence of H 2 O 2 as a substrate for PO, and with o-dianisidine only as a substrate for oxidase (sees Section 2.4). The Cu/Ce S specificity to H 2 O 2 is the important valuable characteristic for applying it as a selective PO-mimetic element in sensors and oxidase-based biosensors. Figure 3 shows the effect of quantity on the NZ immobilized on the GE surface, based on the amperometric signal as a response to H 2 O 2 addition. Table 4 presents comparative analytical characteristics of the recently developed metallic NZ-based sensors for H 2 O 2 analysis, including the results described here. Cu/Ce S specificity to H2O2 is the important valuable characteristic for applying it as a selective PO-mimetic element in sensors and oxidase-based biosensors. Figure 3 shows the effect of quantity on the NZ immobilized on the GE surface, based on the amperometric signal as a response to H2O2 addition. Table 4 presents comparative analytical characteristics of the recently developed metallic NZ-based sensors for H2O2 analysis, including the results described here.

Application of Cu/Ce S as a PO-Mimetic in Amperometric Sensors
To demonstrate the applicability of Cu/Ce S as a chemosensor for H 2 O 2 detection, analysis of H 2 O 2 concentration was carried out in samples of commercial products (Figure 4). The disinfectant samples tested were Famidez-Sanosil, Spray Antiseptic for arms and a pharmaceutical solution of 3% H 2 O 2. The standard addition test, SAT, approach was used to avoid undesirable effects on analytical results from additives in the tested solutions.
Graphical SAT is a type of quantitative analysis often used in analytical chemistry when a standard is added directly to the aliquots of the analyzed sample. SAT is used in situations where sample components may contribute to the analytical signal, thus making it impossible to use a routine calibration method. Estimation of H 2 O 2 in the initial sample was performed using the equation C = AN/B, where A and B are parameters of a linear regression and N is the dilution factor.
The results of hydrogen peroxide determination in commercial samples in comparison to data of manufacturers are presented in Table 5. The average H 2 O 2 concentrations determined from the data in Figure 4 correlated well with the manufacturer's data (Table 5), with an error of less than 10%.

Application of Cu/Ce S as a PO-Mimetic in Amperometric Sensors
To demonstrate the applicability of Cu/Ce S as a chemosensor for H2O2 detection, analysis of H2O2 concentration was carried out in samples of commercial products (Figure 4). The disinfectant samples tested were Famidez-Sanosil, Spray Antiseptic for arms and a pharmaceutical solution of 3% H2O2. The standard addition test, SAT, approach was used to avoid undesirable effects on analytical results from additives in the tested solutions.

Conclusions
In the current research, a number of NPs based on transition and noble metals were synthesized by chemical methods and were screened for their ability to decompose hydro-gen peroxide in solutions. Structure, size, morphology, composition, catalytic properties, and electrochemical activities of the chosen PO-like NZs were characterized on the electrode. A more detailed study was performed for Cu/Ce S -NZ, which was found to be the most effective mimetic of PO. It was demonstrated that the synthesized Cu/Ce S -NZ may be successfully used as an artificial PO for sensor analysis of hydrogen peroxide in commercial disinfectant samples.
Informed Consent Statement: "Not applicable" for studies not involving humans. Data Availability Statement: "Not applicable" for studies not involving humans.

Acknowledgments:
We acknowledge Mariya F. Ivash (Institute of Cell Biology, Lviv, Ukraine) for technical support and experimental assistance.

Conflicts of Interest:
The authors declare no conflict of interest.