The study analyses nanotechnology advances in the synthesis of catalytic nanozymes that mimic enzymatic properties, but are also inorganic nanomaterials. It compares Zn-Mn-Cu multioxide and Mn-Cu multioxide nanomaterials in the role of nanozymes and photocatalysts, in the decomposition of trypan blue dye. The study showed that Zn-Mn-Cu multioxide nanoxide has activity as a peroxidase-like nanozyme (100 mUnit/mL) and achieves 75% dye degradation as a photocatalyst under UV light. While Mn-Cu multioxide shows enhanced photocatalysis under visible light (45% degradation), consistent with its biocatalytic hydrolysis mechanism. In addition, both materials showed biocatalytic activity in the degradation of trypan blue dye (25%), without an additional light source. Further investigation of these dual nanozyme activities may reveal new solutions for their environmental use.
Research Article
Multioxide nanomaterials Zn, Cu, Mn: Combining nanozyme and photocatalyst functions for environmental applications
https://doi.org/10.21203/rs.3.rs-4016819/v1
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nanozymes
photocatalysis
multimetallic nanooxides
biocatalysis
bionanotechnology
water remediation
Advances in nanotechnology have paved the way for the synthesis of nanoparticles with adapted properties, including shape, size and surface modifications, thereby affecting their chemical and physical properties (Bogdan et al. 2015). Nanozymes are one type of material with catalytic properties, gaining notable attention from researchers (Qiao et al. 2014; McDonald et al. 2015; Yang et al. 2021a).
Nanozymes are nanomaterials that exhibit properties that mimic enzyme-mediated biocatalysis. Thanks to their physicochemical properties, combining the characteristics of inorganic materials and enzyme-mimicking activity, while preserving the nature of the nanomaterial formula (Huang et al. 2019). Natural enzymes are often limited by their thermostability and activity in a specific pH range, making them vulnerable to environmental changes (Wong et al. 2021). On the contrary, nanozymes can operate effectively outside the typical physiological parameters of temperature and environmental pH, without denaturing or significantly impairing their catalytic activity. This wider range of stability and enzyme-mimicking functionality make nanozymes an alternative between photocatalytic nanomaterials and systems with immobilised proteins (Wang et al. 2019; Yang et al. 2021b).
Nanozymes have the ability to mimic natural enzymes, allowing them to reduce reactive oxygen species (Liu et al. 2021; Yang et al. 2022), whether to reduce the effects of oxidative stress on cells (Singh et al. 2021). Currently known nanozymes mimic the activities of enzymes from the oxidase or hydrolase groups (Huang et al. 2019). In particular, nanozymes with the character of enzymes from oxidoreductase groups such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) or glutathione peroxidase (GPX) are distinguished (McDonald et al. 2015; Pietrzak and Ivanova 2021). An important element in defining a material as a nanozyme is the determination of the catalytic activity, which should result from mechanisms similar to those of enzymes (Zhang et al. 2022a).
Nanozymes can be tailored for specific reactions, enabling their use as biosensors (Zhang et al. 2022b) and in other biomedical applications such as bioimaging (Sharma et al. 2019; Chen et al. 2021). Currently, nanozymes are being investigated as reactants for the detection of heavy metals (Huang et al. 2019) or catalysts (Huang et al. 2021), and their use for environmental applications is only being developed (Meng et al. 2020; Wong et al. 2021). One environmental application is the use of nanomaterials as photocatalysts in decomposition reactions of dyes and organic substances (Martínez-Vargas et al. 2019; Wang et al. 2021). Photocatalyst materials based on copper oxides (Karthik et al. 2022), zinc oxides (Martínez-Vargas et al. 2019) or manganese oxides (Zohra et al. 2023), can be found in the literature, but trioxide systems of such nanomaterials with enzymatic activity have not been widely studied for use in photocatalysis. Multi-oxide materials, in studies of nanozymes, are most often used as glucose sensors, and studies of their activity are not aimed at using biocatalysis (Han et al. 2018). Comprehensive studies on the enzymatic reaction mechanisms of nanozymes and studies in the use of nanozymes in the dual role of photo- and biocatalysts are lacking in the literature.
The aim of the present study was to obtain and compare the activity of Zn-Mn-Cu and Mn-Cu multioxide nanomaterials with dual nanozymatic and photocatalytic properties. The materials were characterised in terms of their enzymatic properties and their activity in the photocatalytic decomposition of the aqueous model pollutant trypan blue. In order to investigate their enzymatic activity, the activity towards the ABTS radical was determined, proteolytic assays were performed with casein degradation and peroxidase activity in H2O2 decomposition was determined - and then a possible biocatalysis mechanism was proposed for each nanoxide, respectively. The materials contained manganese and copper oxides, due to the presence of these elements in the active centres of the enzymes. Investigations under ultraviolet light and under light mimicking visible light allowed comparison of the photocatalytic activity of the nanomaterials. Biocatalysis studies demonstrated the usefulness of the materials in decomposing trypan blue based on the enzymatic nature of the nanomaterials.
Copper (II) sulphate pentahydrate CuSO4•5H2O (Sigma Aldrich), zinc (II) chloride ZnCl2 (Sigma Aldrich) and manganese (II) sulphate monohydrate MnSO4•H2O (Sigma Aldrich) were used as oxide nanoparticle precursors. Sodium hydroxide NaOH (Sigma Aldrich) was used as precipitating agent. The enzymes used as reference material was peroxidase from horseradish (Sigma Aldrich) and bromelain from stem (ThermoScientific), additionally pineapple extract from cosmetic company was sued as an enzymatic reference (ZróbSobieKrem – “ZSK”). Materials used in activity testes and sorption were: trichloroaceticacid (TCA) (Sigma Aldrich), 6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxilic acid (TROLOX) (Sigma Aldrich), Folin–Ciocâlteu phenolic reagent (sigma Aldrich), casein (Sigma Aldrich), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Sigma Aldrich), albumin from bovine (Sigma Aldrich), hydrogen peroxide (POCH), guaiacol (Sigma Aldrich), trypan blue (Sigma Aldrich).
3.1. Synthesis of nanomaterials
A co-precipitation method was used to obtain the nanooxides, the multimetallic nanooxides were labelled as 'Zn-Mn-Cu multioxide' and 'Mn-Cu multioxide'. The synthesis was carried out in a MAGNUM II microwave reactor, in a teflon vessel. Aqueous solutions of copper (II) sulphate, manganese (II) sulphate and zinc (II) chloride and a solution of NaOH as a precipitating agent were prepared. Zinc (II) chloride solution (C = 3.5 mol/L, V = 4.82 mL) was added directly to the teflon vessel and precipitated with NaOH solution (C = 3 mol/L, V = 14.52 mL). Then, copper sulphate (C = 0.62 mol/L, V = 5.69 mL), manganese sulphate (C = 0.96 mol/L, V = 6.00mL) solutions were dropped simultaneously while stirring in a Hielscher UP400St sonicator. The systems were homogenised for 5 minutes.
Similarly, bioxide material was prepared, where solutions of copper sulphate (C = 1 mol/L, V = 6.00mL) and manganese sulphate (C = 3.5 mol/L, V = 4.82mL) were added directly to the teflon vessel and precipitated with NaOH solution (C = 3 mol/L, V = 14.52mL) while simultaneously stirring in a Hielscher UP400St sonicator. The systems were homogenised for 5 minutes.
The vessel with a total volume of 30 mL was then placed in a MAGNUM II microwave reactor and the process was carried out at t = 10 min, T = 180°C and p = 20 bar. After the process, the material was filtered on 0.2 µm nitrocellulose strainers on a vacuum set and then dried for 24 h at 70° C. After drying, the material was crushed in an agate mortar.
3.1.1. Characterisation of nanomaterials
The morphology of the resulting metal oxide compounds was investigated by scanning electron microscopy with energy dispersive spectroscopy (Hitachi TM 3000).
The crystallites of the nanoparticles were analysed by structural roentgenography (XRD) using a Philips X'Pert Camera diffractometer with a PW 1752/00 CuKa monochromator in the 2θ angle range, from 10 to 80°. In addition, the size of the crystallites was calculated from the Scherrer equation:
$$\text{d}=\frac{\text{K}\bullet {\lambda }}{\text{F}\text{W}\text{H}\text{M}\bullet \text{cos}{\theta }}$$
Where: d – average size of crystallites, FWHM – peak width at half of its height, proportional to the size of the crystallite, K – Scherrer’s constant, λ – X-ray wavelength, θ – angle formed by radiation with the atomic plane. The constant K was selected based on shape of particle as 0,89.
3.2. Determination of enzymatic activity of nanomaterials
Following the physico-chemical characterisation of the materials, enzymatic activity determination methods were used to confirm the nanozymatic activity of the materials tested. The materials were tested for proteolytic activity (protein chains cleavage) - which verified the ability to enzymatically hydrolyse environmental contaminants. Anti-radical activity with the ABTS radical, determined the ability to reduce the radicals formed during decomposition. The enzymatic activity of nanomaterials mimicking the activity of peroxidases used the reduction reaction of H2O2 with the simultaneous oxidation of guaiacol - verifying this activity provided an insight of the possible enzymatic reaction mechanism of nanozymes in the oxidoreduction of pollutants.
3.2.1. ABTS method - antiradical activity
The ABTS method measures spectrophotometrically the reduction of the ABTS + radical, using TROLOX as a reference substance. The ABTS + radical is generated by 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid. This radical is a chemically stable chromophore compound with a wide pH range, which is soluble in water and shows strong absorption in the 600–750 nm range. The antioxidants then retain the ABTS + radical [2,2-azino-bis(ethylbenzenothiazoline-6-sulfonic acid)], leading to a decrease in absorbance, which is detected by the antioxidant-radical combination at different times (Miller et al., 1993).
A solution of ABTS (6mM) was prepared with potassium persulphate (2.45mM) in water, the solution was left in the darkroom with an ageing time of 16 h. A sample (0.1 mL of enzyme or 10mg of nanomaterial) was added to 4 mL of ABTS reagent, incubated for 10 min and then measured at 734 nm. In the assay, the nanomaterials were compared to the enzymes peroxidase and bromelain, which were diluted to similar activity. The calibration curve was based on the reaction of Trolox with ABTS reagent, the measured absorbance after the reaction corresponds to the amount of Trolox needed for the reaction expressed in µmol/mg. The curve was prepared in the range 0-40-80-120-160-200 µmol TROLOX, and the concentration was calculated from the formula:
$$y = -\text{956,05}x + \text{378,95}$$
$$R² = \text{0,9919}$$
Where: y – absorbance, x – TROLOX concentration (µmol)
3.2.2. Casein method - proteolytic activity
Casein was used as a substrate to measure the proteolytic activity of the materials and materials after immobilisation. During hydrolysis of the casein protein, the reaction products are precipitated with TCA acid and then determined colourimetrically with a Folin solution measuring the concentration of the reaction products. The calibration curve of the method is based on tyrosine, which is released during protein degradation.
A casein solution (5 mL) was added to the test sample (10 mg) and the reaction was run for 30 minutes at 37°C. The reaction was then stopped with TCA acid (5 mL) and incubated for a further 30 minutes. The filtrate was collected and labelled with Folin solution (2 mL), incubating the mixture for 30 minutes. In the final step, the filtrate was collected and its absorbance measured spectrophotometrically at 726 nm. As reference samples, commercially available pineapple extract (labelled ZSK) and pure bromelain were used - both the extract and the pure enzyme exhibit proteolytic activity.
The calibration curve was determined in the range of 0–0.05–0.1–0.2–0.4–0.5 tyrosine concentrations. From the curve formula, the amount of released tryosine (x) was calculated from the absorbance reading (y):
$$y = \text{1,1595}x - \text{0,0214}$$
$$R² = \text{0,9977}$$
Where: y - read absorbance, x - amount of tyrosine released (µmol).
The result was converted by the volume of the reaction mixture (Vt = 11 mL) and time (t = 10min), the volume of enzyme/sample (Vs = 1 mL) and the volume after filtration (Vf = 2 mL) obtaining the final result expressed as unit/mL. The following formula was used:
$$\frac{unit}{ml}=\frac{x \times {V}_{t} }{t\times {V}_{s}\times {V}_{f}}$$
3.2.3. Peroxidase activity
Peroxidase breaks down H2O2 by converting it to water, with simultaneous oxidation of the reaction substrate. The peroxidase reaction can be monitored using guaiacol (2-methoxyphenol), which can be oxidised to produce a brown product (tetraguaiacol), which is quantified using a spectrophotometer at 420 nm. A calibration curve was prepared using peroxidase to a concentration of 0-0.1-0.5-1-1.5 U/mL, and the concentration was calculated from the formula:
$$y = -\text{0,0697}{x}^{2} + \text{0,2228}x + \text{0,0074}$$
$$R² = \text{0,9991}$$
Peroxidase activity was measured by the reaction of H2O2 decomposition (12mM, 0.03mL) by the enzyme with guaiacol (20mM, 0.05mL) as an indicator to change the colour of the solution. 0.1 mL of enzyme or 10 mg of sample was added to the mixture and then, after 10 minutes of reaction, the absorbance of the solution was measured.
3.3. Studies on dye degradation in photocatalysis and biocatalysis
3.3.1. Photocatalytic decomposition - UV and Vis-LED
Photocatalytic studies for nanozymes were carried out using trypan blue (TB) dye at a concentration of 50ppm. The photocatalytic activity of the tested materials was evaluated by monitoring their ability to degrade the trypan blue dye in an aqueous environment in the presence of UV ultraviolet light (wavelength 364 nm, output power 12.2W) and artificial visible LED light (intensity of light emitted 2750 lumen, output power 36W). The degradation reaction was carried out at room temperature, for 60 min, sampling every 15 min. The calibration curve was based on the measurement of TB absorbance at 590nm, in the range 0-10-20-30-40-50 ppm, and the concentration was calculated from the formula:
\(y = \text{0,035}x + \text{0,0481}\) \(R² = \text{0,9994}\)
Where: y - absorbance, x - TB concentration (mg/L)
The dye solution (5 mL) was added to the test material samples (5 mg) and stirred for 30 minutes in a darkroom to achieve adsorption and desorption equilibrium. The reaction mixture was then exposed to ultraviolet and visible light to study the photocatalytic degradation of the dye in the presence of Zn-Mn-Cu multioxide and Mn-Cu multioxide materials. After a set time (5-10-15-30-45-60 min), the material was filtered on a syringe filter (0.22µm) and the dye concentration was monitored spectrophotometrically. The tests were carried out in duplicate. The solution of the dye itself (blank) was also photodegraded, in parallel with the test samples, to exclude the effect of decomposition of the dye itself. The result for the material in a given time unit was related to the parallel blank.
In addition, tests for multiple applications of nanomaterials in the decomposition of the dye were carried out, repeating the photocatalysis reaction three times. For the cycling reaction, samples were prepared as for the UV-induced photocatalytic activity test − 5 mL of dye and 5 mg of material were used. Each catalytic cycle lasted 15 min, after a given cycle the nanomaterial and dye were separated by centrifugation. After 10 min of centrifugation (10,000 rpm), the dye was collected for absorbance testing and the material used was purified with distilled water (2mL) and separated by centrifugation before the next cycle. A new batch of dye was added to the material thus purified.
To evaluate the performance of the cycles, the equation was used:
$$\text{D}\text{e}\text{g}\text{r}\text{a}\text{d}\text{a}\text{t}\text{i}\text{o}\text{n} \text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}\text{y}=\left(\frac{{C}_{t}}{{\text{C}}_{0}}\right)$$
Where: C0 and Ct are the respective dye concentrations at t = 0 (initial time) and t = 15 min.
3.3.2. Biocatalytic decomposition
Biocatalytic tests were carried out using trypan blue (TB) dye and the addition of H2O2. The study was designed to investigate the effect of enzymatic degradation of the dye by the materials and to check the enzymatic mechanism of the reaction. The degradation reaction was carried out at room temperature. The calibration curve was based on the measurement of TB absorbance at 590 nm, in the range 0-10-20-30-40-50 ppm, and the concentration was calculated from the formula:
\(y = \text{0,035}x + \text{0,0481}\) \(R² = \text{0,9994}\)
Where: y - absorbance, x - TB concentration (mg/L)
As in photocatalysis, the same methodology was used, a dye solution (5ml) was added to a sample of material (5mg) at an initial concentration of 50ppm and H2O2 (0.042%, 0.25mL) was added and the reaction was run for a set time (5-10-15-30-45-60 min), then the material was filtered on a syringe filter (0.22µm) and the dye concentration was monitored spectrophotometrically. The study was carried out in duplicate. To evaluate the degradation efficiency, the equation used was:
where C0 and Ct are the respective dye concentrations at t = 0 (initial time) and t = set time [min].
4.1. Materials structural analysis
The XRD analysis of the materials confirmed the presence of oxide phases suitable for each of the materials (Fig. 1).
In the Zn-Mn-Cu multioxide material, ZnO (31.8°, 34.46°, 36.28°, 47.56°, 56.62°, 62.88°, 66.38°, 67.98° and 68.1°) and Cu Mn2O4 (18.4°, 30.1°, 35.48°, 53.84°, 57.5°, 62.88°, 66.38°) phases are present. These phases are reflected in the COD crystallographic data #2300116 and #1533674, respectively, for the phases. The phase ratio was 1:0.21, the elemental molar ratio was assumed as 1:0.2:0.3 and finally determined as 1:0.07:0.12 (Zn:Cu:Mn).
The Mn-Cu multioxide material contains Mn3O4 (18.01°, 28.9°, 38.1°, 44.4°, 53.9°, 56.1°), CuO (32.4°, 38.6°, 48.7°, 58.5) and Cu2O (42.32°, 61.4°, 69.7°) phases, corresponding to records with the respective COD numbers #1011262, #9016105 and #1010941. The oxide phase ratio was 1:0.125:0.06. The theoretical elemental molar ratio (Mn:Cu) was expected to be 1:0.35, which was verified as 1:0.23 after XRD analysis.
Material sizes were calculated from XRD data - average crystallite phase sizes were included in the Table 1, for each phase of nanomaterials.
| Material | Phase | Size [nm] |
|---|---|---|
| Zn-Mn-Cu multioxide | ZnO | 50,25 |
| CuMn2O4 | 27,70 | |
| Mn-Cu multioxide | Mn3O4 | 18,08 |
| CuO | 7,85 | |
| Cu2O | 5,97 |
For the Zn-Mn-Cu multioxide material, the XRD results obtained for the ZnO phase are in agreement with those presented by the Raha et al. (Raha et al. 2021).
The researchers synthesised a CuO/Mn3O4/ZnO nanomaterial system - using the precipitation method to first obtain copper oxide, followed by zinc oxide and manganese oxide (II/III). The material was placed in an autoclave for the reaction and then the material was further calcined. In addition, the researchers characterised the different phases of the system. The size of the ZnO phase is larger than in study of Raha et al. (Raha et al. 2021), however, this may be due to the different order in which the oxides were obtained and the different methodology for synthesising the trioxide. The spinel phase of CuMn2O4 is consistent with the XRD results presented by Sanjabi S et al. (Sheikhzadeh and Sanjabi 2021) on copper manganese oxide films obtained by co-electrodeposition. The authors of the study particularly highlighted the presence of a peak at 18.4°, which determines the presence of the CuMn2O4 phase.
For the Mn-Cu multioxide material, the spectra for the CuO and Mn3O4 phases are also found to be similar in the study of Raha et al. (Raha et al. 2021), and the crystallite sizes are also similar – 10 nm for the CuO phase and 14 nm for the Mn3O4 phase. Similarly, in the study of Lee H et al. (Shaislamov et al. 2016), who obtained nanorods with CuO as the base doped with ZnO nanoxide. The authors prepared CuO films using copper (II) sulphate and sulphuric acid (VI), which was oxidised at 300°C for 2h. Then, by repeating the oxidation of zinc acetate to ZnO on the CuO surface several times, the researchers applied a layer of zinc oxide to the resulting film and the system thus prepared was reacted in a hydrothermal process. The researchers presented XRD spectra containing the different phases: CuO, Cu2O and ZnO, which can be compared with the phases obtained for Zn-Mn-Cu multioxide and Mn-Cu multioxide materials.
In the SEM microphotographs (Fig. 2) the difference in material structures can be observed. The Zn-Mn-Cu multioxide contains larger, single nanocomposite particles, on which smaller particles can be seen, forming a differentiated material structure. A difference in the shape of the nanoparticles can also be observed, with the larger elements being flat and asymmetrical and the smaller ones being more circular. The EDS analysis shows an even distribution of the zinc phase on the surface of the material, apart from the areas occupied by manganese and copper. It can be seen that Mn and Cu are visible in the same areas, confirming that the elements occur together. The Mn-Cu multioxide morphology shows more agglomerated particles of similar size with a swollen structure. The nanomaterial contains rather round structures and particle layering is visible. The microphotographs from the EDS analysis show an even distribution of both elements, manganese and copper, on the surface of the material. It is also possible to see areas occupied only by copper, which is evidence of phase separation.
Similar characteristics can be seen for the materials obtained by the team of Mohamed M et al. (Mohamed et al. 2023), who prepared Mn3O4/ZnO and CuO/ZnO-containing materials from chloride precursors by co-precipitation using a hydrothermal process.
4.2. Determination of enzymatic activity of nanomaterials
Biocatalytic assays were performed to investigate the enzymatic activity of the nanomaterials tested as potential nanozymes. The nanomaterials were compared with two groups of enzymes: hydrolases (bromelain) and oxidoreductases (peroxidase). The mechanism of action of these biocatalysts is different, so the appropriate choice of bioactivity assays allowed conclusions to be drawn about the potential mechanism of action of the nanozymes. The results helped to assign them a potential enzymatic reaction mechanism and identify different biocatalytic behaviours.
4.2.1. ABTS method - antiradical activity
Each of the synthesised materials showed anti-radical activity against the ABTS radical, which could be compared with the results for the pure enzymes peroxidase and bromelain (Fig. 3). The anti-radical activity of the Zn-Mn-Cu multioxide material could be compared with that of peroxidase, and that of the Mn-Cu multioxide material with that of bromelain. Compared to the homogeneous CuO and MnO oxides synthesised by Neethidevan et al. (Neethidevan et al. 2023), Zn-Mn-Cu multioxide and Mn-Cu multioxide show twice the activity against the radical. In addition, Zn-Mn-Cu multioxide shows similar activity to CuO in study of Rehana et al. (Rehana et al. 2017), and Mn-Cu multioxide twice this value.
4.2.1. Casein method - proteolytic activity
The proteolytic activity assay (Fig. 4) was used to determine the direction of contaminant degradation by nanomaterials - according to the mechanism of enzymatic hydrolysis. Bromelain and the extract containing this enzyme belong to the group of hydrolases - enzymes that degrade the substrate by hydrolysis. These substances were used as a reference to measure the activity of the materials. Zn-Mn-Cu multioxide did not show proteolytic activity, due to the presence of zinc. Mn-Cu multioxide probably due to its higher manganese oxide content showed activity at 0.2 U/mL. The pure bromelain enzyme showed an activity of 0.45 U/mL. The commercial extract showed an activity of 0.25U/mL, approximately 48% relative to pure bromelain. The Mn-Cu multioxide material was capable of degrading the protein in a hydrolysis reaction.
According to Kiewiet et al. (Kiewiet et al. 2018), the reaction catalysed by enzymes from the hydrolase group can be represented as in the following equation:
$$R-CO-NH-R+ HO-H \underrightarrow{enzyme} R-CO-OH+H-NH-R$$
4.2.2. Peroxidase activity
Further analysis was followed by a peroxidase activity assay (Fig. 5) involving the oxidation of guaiacol in the presence of hydrogen peroxide. The activity of Zn-Mn-Cu multioxide was twice as high as that of the Mn-Cu multioxide material - it was 0.1 U/mL versus 0.04 U/mL. In a study by Alzahrani et al. (Aldhahri et al. 2021), the activity of immobilised horseradish peroxidase on CuO on SDS polymer matrix was 0.2 U/mg, with 2% Cu addition.
Enzymes of the peroxidase group facilitate electron transfer from the donor molecule to hydrogen peroxide, leading to the oxidation of the donor molecule and the reduction of hydrogen peroxide to water. According to Oliveira et al. (Kerstner De Oliveira et al. 2021), the electron transfer occurs at the active centre of the enzyme and, importantly, two electrons are generated during the reaction - unlike the Fenton reaction - after which the enzyme returns to the ground state. The reaction can be schematically represented as:
$$ROOH+A{H}_{2} \underrightarrow{enzyme} {H}_{2}O+ROH+A$$
Where: A - electrone donor in the oxidized form
or as a decomposition of H2O2 if no other substrate is present:
$${2H}_{2}{O}_{2}\underrightarrow{enzyme}2{H}_{2}O+{O}_{2}$$
4.3. Studies on the photocatalytic and biocatalytic activity of nanomaterials
After investigating possible reaction mechanisms, the photocatalytic properties of the nanomaterials were examined. The decomposition efficiency of the trypan blue dye in the presence of UV-light materials and under Vis-LED light was compared. Then, combining enzymatic activities in the catalysis, the decomposition of the dye in the presence of H2O2 was carried out - verifying the applicability of nanomaterials as nanozymes for the decomposition of environmental pollutants.
4.3.1. Photocatalytic decomposition
Photocatalytic activity was tested after 30 min of dye sorption in the dark conditions and then the TB decomposition for each material was measured at 15 min intervals (Fig. 6). The decomposition by material Zn-Mn-Cu multioxide exceeded 70% and by material Mn-Cu multioxide 50%, after one hour of light irradiation. For the reference material ZnO, the decomposition was over 90%. The results for these materials are higher than for the ZnO-Tb material used by Ghrib et al. (48%) (Ghrib et al. 2021). However, according to the study by Migdadi et al. (Migdadi et al. 2022) for the ZnO/CuO material, containing 10% mas. copper addition, the photocatalysis of the dye reached 97% after 90 minutes of irradiation, which could suggest an extended irradiation process.
The materials were also examined under Vis-LED light imitating sunlight (Fig. 7) to test the feasibility of the nanomaterials in decomposing the dye in an aqueous environment without the use of an artificial light source. The Mn-Cu multioxide material showed the best activity under Vis-LED light, reducing trypan blue by more than 45%. The material Zn-Mn-Cu multioxide and pure ZnO oxide achieved a dye reduction of 38% and 32%, respectively. In the literature, materials of CuO doped with zinc or iron can be found, resulting in approximately 40% decomposition of the MB dye under visible light mimicked by a xenon light source, developed by George et al. (George et al. 2022).
In addition, the materials were tested in repeated 15-min photocatalytic cycles under UV light Table 2). In the first cycle, Zn-Mn-Cu multioxide could be compared with ZnO reducing the amount of dye by 60%, Mn-Cu multioxide by 47%. In the next two cycles, the activity of materials Zn-Mn-Cu multioxide and Mn-Cu multioxide remained at a similar level degrading the dye by about 20% of the initial value. The activity of ZnO also decreased, however, it retained more activity than the nanozymes. Compared to the material consisting of Fe2O3 obtained by the team of Fattahi et al. (Fattahi et al. 2023), the cyclic use of nanomaterials also showed a decrease in catalytic activity by nearly half (74–47%, over two cycles). According to the authors of the study, this is a negative feature that should be moderated in future studies.
|
Material |
Zn-nanoxide |
Mn-nanoxide |
ZnO |
|---|---|---|---|
|
Cycle 1 |
64% |
47% |
62% |
|
Cycle 2 |
22% |
21% |
48% |
|
Cycle 3 |
20% |
18% |
40% |
4.3.2. Biocatalytic decomposition
The next stage was to use enzymatic activity to decompose the TB dye in the presence of hydrogen peroxide, as shown in Fig. 8. For the materials tested, the addition of peroxide resulted in decomposition of the dye by 24% for the Zn-Mn-Cu multioxide material and 27% for the Mn-Cu multioxide. An important element in the study was the absence of an additional light source to affect activity. The biocatalytic activity achieved is only due to the enzyme-like behaviour of the nanomaterials. In the study by Janović et al. (Janović et al. 2017), the degradation of azo dye (RB52) by the enzyme horseradish peroxidase immobilised on chitosan material was carried out. The activity of the system was also compared with the pure, non-immobilised HRP enzyme. In the experiments, H2O2 was added from 0.22mM to 4.4mM. The decomposition of the dye (RB52) after 4h for the pure enzyme was 20%. Compared to the nanozymes tested in the current research, the HRP enzyme mediated decomposition took significantly longer, despite the same final degradation result.
The addition of H2O2 enabled enzymatic decomposition of the dye according to the reaction:
$$ROOH+A{H}_{2} \underrightarrow{enzyme} {H}_{2}O+ROH+A$$
for material Zn-Mn-Cu multioxide
$$R-CO-NH-R+ HO-H \underrightarrow{enzyme} R-CO-OH+H-NH-R$$
for material Mn-Cu multioxide
In the present study, Zn-Mn-Cu multioxide and Mn-Cu multioxide nanomaterials were synthesised and characterised as materials combining the properties of nanozymes and photocatalysts, and mechanisms for the degradation of trypan blue dye were proposed. Using the ABTS radical reduction reaction and comparing the activity of the materials to pure enzymes, it was possible to determine the initial similarity of the nanomaterials to selected biocatalysts with antiradical activity. Determination of proteolytic activity levels allowed the determination of mechanisms for the degradation of aqueous contaminants through the use of hydrolysis. The peroxidase activity allowed further characterisation of the nanomaterials, bringing conclusions on the bioactivity of the nanozymes closer. The selection of biocatalytic methods allowed the determination of the similarity and association of the nanomaterial activity with the corresponding enzyme group of hydrolases (Mn-Cu multioxide) or oxidoreductases (Zn-Mn-Cu multioxide), as well as an approximation of the reaction mechanism of the biocatalytic degradation of trypan blue dye.
Zn-Mn-Cu multioxide appears to be a versatile nanomaterial with nanozyme and photocatalytic activity, due to the simultaneous presence of a zinc oxide phase used for photocatalysis and a CuMn2O4 spinel phase, two metals found as active centres for enzymes (Schmidt and Husted 2019; Alizadeh and Salimi 2021). The activity of the Zn-Mn-Cu multioxide material was similar to that of the peroxidase enzyme, as confirmed by the activity towards the ABTS radical (218.6 µmol/ml for the nanozyme and 259.8 µmol/ml for the pure enzyme). The peroxidase activity for Zn-Mn-Cu multioxide was twice that of Mn-Cu multioxide, at 100 mUnit/ml. The materials resembled enzymes from the oxidoreductase (Zn-Mn-Cu multioxide) or hydrolase (Mn-Cu multioxide) groups, respectively, to propose a biocatalytic mechanism for the decomposition of the dye (Kerstner De Oliveira et al. 2021). The photocatalytic activity under ultraviolet light for Zn-Mn-Cu multioxide was 20% higher than Mn-Cu multioxide, due to the presence of zinc. Also under LED light mimicking visible light, the zinc containing material was active due to the presence of copper and manganese. As reported by Ebrahimi et al. (Ebrahimi et al. 2019), doping zinc oxide with other metals allows to reduce the energy gap in the oxide structure, thus broadening the absorption spectrum from ultraviolet to visible light. Nevertheless, Mn-Cu multioxide exhibited higher photocatalytic activity under vis-LED light, achieving 46% degradation of trypan blue. Biocatalysis studies using H2O2 as substrate did not show significant differences between the materials, achieving approximately 25% degradation of the initial dye concentration. Due to the higher antiradical activity and different mechanism of action of Mn-Cu multioxide, the biocatalysis of the dye may have first reduced H2O2 and then used the resulting water to further hydrolyse the dye (Kiewiet et al. 2018). However, the Mn-Cu multioxide nanomaterial also showed catalytic activity under UV light and activity as a nanozyme, which does not exclude its use in photocatalysis.
Further studies using the dual activities of nanozymes may bring more solutions closer in photobiocatalysis of environmental pollutants.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Competing Interests
The authors have no relevant financial or non-financial interests to disclose.
Author contribution
Conceptualization: [Julia Matysik, Marcin Banach]; Methodology: [Julia Matysik, Olga Długosz]; Formal analysis and investigation: [Julia Matysik]; Writing - original draft preparation: [Julia Matysik]; Writing - review: [Olga Długosz, Marcin Banach], Writing- editing: [Julia Matysik], Supervision: [Marcin Banach]. All authors read and approved the final manuscript.
Data availability
Not applicable or available upon request
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No competing interests reported.