The morphologies and diameters of Cu hNFs synthesized with algae extract were detailed by FE-SEM analysis, elemental composition of NFs by EDX mapping results, and functional groups were detailed by FT-IR analysis. The peroxidase-like catalytic activities of hNFs have been explained by a Fenton-like mechanism.
3.1 Characterization of hNFs
According to the characterization test results, the diameters of hNFs and petals synthesized as a result of the reaction of 1 ml A. mirabilis extract and 8x10-4 M Cu ion in 10 mM PBS buffer (pH: 7.4) were 31 µm (Figure 1a), and 27 nm (mean), respectively. (Figure 1b). With the change in the concentration of the plant extract, and the pH of the PBS medium, disruption in the morphological structures of the synthesized hNFs and differences in the size distribution were determined (Figure 2a-d). It was observed that no blue precipitate was formed and synthesis did not occur in the tubes (at all plant concentrations) under pH 5 conditions of PBS. The formation mechanism of NFs has been detailed in the previous literature [4, 9, 10]. In the mechanism mainly consisting of nucleation, growth and finish phase, the process that starts with the formation of primary phosphate crystals as a result of the reaction of Cu ions and amide, hydroxyl and diol groups of the bioextract (nucleation phase) is completed with the arrangement of the petals [4, 9, 10, 15, 16]. Baldemir et al., 2020 determined the diameters of hNFs synthesized with Artemisia absinthium, A. vulgaris, and A. ludoviciana extracts in the range of 2-10 μm . In addition, it has been reported that while hNF is synthesized with the use of 0.1 mg/ml bioextract in the reaction, hNF formation was not observed with the use of 0.5 mg/ml bioextract. In their studies, the researchers showed that the bioextract content and concentration as an organic component had a significant effect on the formation and size of hNF. In another study, NF synthesis did not occur at 0.02, 0.03, and 0.05 mg/ml concentrations of Trigonella foenum-graecum extract; in addition, it was emphasized that the encapsulation yields of NFs increased by increasing the concentration of the extract from 0.1 mg/ml to 0.5 mg/ml . Guven et al., (2021) reported that Cu-based hNFs synthesized with cheery stalk extract were synthesized in the pH 6-9 range of PBS buffer, while NFs were not synthesized in other pH conditions . In a study in which urease-based NF was synthesized in PBS conditions in the pH range of 6-9, this was explained by the effect of medium pH on the binding affinity of urease molecules and Cu ion . Although an interesting study reported that chance is an important factor in the growth of NF , on the contrary, we claim that concentration of extracts, and pH of PBS significantly affects the size, morphology and formation of NFs based on the consistency of our findings and literature reviews.
Inorganic and organic components of hNFs determined by using EDX (Figure 3) and FT-IR (Figure 4) analysis, respectively. The presence of Cu and other components in the structure of NFs is demonstrated by EDX spectrum (Figure 3a) and EDX mapping (Figure 3b-f). The weight % of Cu was determined at %15.45 in hNF. The distribution of four key elements including C (turquoise color), O (green color), P (yellow color), and Cu (red color) in NF was proved with EDX mapping (Figure 3 b). The elements of C (Figure 3c), O (Figure 3 d), P (Figure 3e), and Cu (Figure 3f) analyzed with mapping by representing different color in NF. The functional groups were determined by FT-IR analysis. The presence of C-H (alkane groups) were reveal at 2916 cm-1, 2848 cm-1, and 1453 cm-1 wavenumber. The peaks at 1652 cm-1, and 1143 cm-1 correspond to amine (-NH), aliphatic ether (C-O), respectively. Primary phosphate crystals formed in PBS buffer were associated with peaks at 1039 cm-1, 987 cm-1, 717 cm-1, 623 cm-1, and 558 cm-1 [4, 9, 19]. Characterization peaks confirmed the formation of organic/inorganic hybrid NFs in PBS buffer along with their morphology.
3.2 Catalytic Activiy of NFs
The peroxidase-like catalytic activity of hNFs were determined by spectrophotometry readings of the oxidation of guaiacol (Figure 5a). The conversion of guaiacol to 3,3-dimethoxy-4,4-diphenoquinone as a result of the reaction is explained by Fenton's mechanism (Figure 5b) [4, 19].
The free radicals formed by the reaction of Cu+1 with H2O2, formed by the reaction of H2O2 and Cu+2 (contained of Cu hNFs) in the reaction medium, provide the oxidation of the substrate (Figure 5b). By this mechanism, 3,3-dimethoxy-4,4-diphenoquinone was formed by the oxidation of guaiacol . Similar to our study, the peroxidase-like catalytic activities of cherry stalk, thymol, allicin, Viburum opulus, and Laurocerausus officinalis-based Cu NFs against guaiacol were explained by a Fenton-like mechanism [4, 9, 19, 20, 21]. Mei et al. (2022) reported that tetracycline degradation caused by CeO2 hNFs is mediated by radicals formed as a result of Fenton's mechanism . Dadi et al. (2020) reported that peroxidase activities of gallic acid@Cu NFs depend on reaction time, substrate concentration and NF morphology . Jiang et al. (2021) explained the peroxidase activity that provides the oxidation of 3,3′,5,5′-tetramethylbenzidine of amino acid-based Cu hNFs with the Fenton mechanism . In the light of this information, we attribute the peroxidase-like catalytic activity of hNFs synthesized with the cooperation of algae extract and Cu to the decomposition of guaiacol by free radicals formed as a result of Fenton's mechanism.
3.3 Antioxidant activity of NFs
Studies to determine the antioxidant activities of nanoparticles and hNFs synthesized by biological method have an important position . Free radicals that occur as a result of various bioreactions and cause oxidative damage are detoxified by antioxidants that prevent the oxidation of molecules [4, 25]. The DPPH scavenging activity with the concentration increase of Cu hNFs is given in Figure 6.
In this study, antioxidant activity of algae@Cu hNFs determined as 50% inhibitory concentration (IC50) were calculated at 2.07 mg/ml. In previous studies, it was noted that the free radical scavenging activity increased with the increase in the concentration synthesized by the biosynthesized nanomaterials [25, 26, 27, 28, 29]. Guven at al., (2022) reported that NFs exhibit enhanced antioxidant activity against DPPH with increasing concentration (IC50: 1.35 mg/ml) . Consistent with previous studies, according to our findings, Cu hNFs showed antioxidant properties by exhibiting DPPH scavenging activity with increasing concentration.