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Synthesizing hybrid copper phosphate (Cu3(PO4)2) nanoflowers using Cu+2 and shed snakeskin: antioxidant, antibacterial, anticancer, guaiacol, anionic, and cationic dye degradation properties

Journal of Biological Engineering volume 19, Article number: 1 (2025) Cite this article

Abstract

Background

Synthesis of organic@inorganic hNFs is achieved by the coordination of organic compounds containing amine, amide, and diol groups with bivalent metals. The use of bio-extracts containing these functional groups instead of expensive organic inputs such as DNA, enzymes, and protein creates advantages in terms of cost and applicability. In this study, the application potentials (antioxidant, antibacterial, anticancer, guaiacol, anionic, and cationic dye degradation) of hybrid (organic@inorganic) nanoflowers (hNFs) synthesized with Cu+2 and snakeskin (SSS) were proposed.

Results

Morphology, presence, and composition of elements of Cu and SSS-coordinated hNFs (Cu@SSS hNFs) were shown through FE-SEM–EDX spectroscopy. According to FE-SEM findings, hNFs synthesized with 0.5 ml and 1 ml extract have diameters of 12.81 and 3 µm, respectively. Diffraction peaks of hNFs determined by XRD were consistent with JCPDS Card 00–022 –0548. Cu@SSS NFs showed antioxidant properties depending on time through DPPH scavenging behavior (ability (R2: 0.5612, IC50: 2.07 mg/ml). Cu@SSS hNFs synthesized coordination of SSS and Cu degraded (75%) methylene blue at the highest pH 9 condition. However, hNFs highest degraded (68%) brilliant blue in an acidic PBS medium. hNFs oxidized guaiacol depending on exposure time. Cu@SSS hNFs demonstrated antibacterial properties towards Gram (-/ +) pathogen strains (MIC: 60 µg/ml). The catalytic and antimicrobial properties of hNFs were mentioned by the Fenton reaction. The cytotoxicity of Cu@SSS hNFs on the lung carcinoma (A549) cell line was shown to be concentration-dependent by the MTT test assay (IC50: 56.4 µg/ml).

Conclusion

As a result, Cu-based hNFs synthesized by using an organic waste (SSS) might be improved for environmental and biomedical applications.

Introduction

Nanomaterials exhibit superior properties with their morphology and properties. For this reason, it has been among the popular topics of both and scientific industrial fields such as electronic, optical, magnetic, nano-radiotracer applications, biomedical fields, enzyme and environmental applications [1,2,3,4,5]. Various metallic NPs, such as monometallic NP and bimetallic NP, are synthesized by different approaches (physical, biological, chemical)—advantageously against each other [6]. In recent years, the biological synthesis method, which is carried out with low cost and energy needs without using chemical reducing agents, has become one of the popular topics [7, 8]. Kiani et al. (2024) reported that ZnO NPs synthesized with Lavandula stoechas extract exhibited antioxidant, anticancer, antimicrobial, and catalytic activities and that ZnO NPs can be used in environmental applications [9]. Zare-Bidaki et al. (2023) reported that AgNPs synthesized with Petroselinum crispum extracts have antifungal, antioxidant, and anticancer properties [10]. It has been reported that Ag NPs synthesized with various biological extracts in particular have application potential in various fields [11, 12]. Barzegarparay et al. (2023) reported that selenium nanoparticles (Se NPs) synthesized with Crataegus monogyna extract exhibited antioxidant and anticancer activity [13]. In another study, researchers reported that palladium (Pd) NPs synthesized with four different plant extracts caused catalytic degradation of thiazine dye (methylene blue), azo dye (methyl orange), and alkaline dye (rhodamine B) [14]. Habib et al. (2022) determined the antimicrobial activities of silver (Ag) NPs synthesized with Zanthoxylum armatum extract [15].

Recently, various nanofibers, magnetic nanoparticles, nanoflowers, and carbon-containing nanostructures have been applied to enrich the stability and catalytic properties of enzymes [16,17,18,19]. Hybrid nanoflowers synthesized by hybridization of organic components containing amine and diol groups with bivalent inorganic components attract attention with their layered surfaces and porous structures. The first time, Zare et al. (2012) synthesized hNFs by hybridization of protein and inorganic Cu+2 inputs and declared that the hNFs obtained by enzyme immobilization exhibited improved activity and stability [20]. A hybrid form of organic and inorganic, hNFs have a flower-shaped and porous structure and they are functional as carriers in the immobilization of biomolecules such as enzymes [16]. The highest surface area (surface/volume) of hNFs gives them an effective loading capacity, enhanced catalytic activity, and high stability. Studies are being carried out on the synthesis and applications of hNFs with various organic components and bivalent ions. Demirbaş et al. (2023a) reported that Cu hNFs, which they synthesized with amino acids (glycine and phenylalanine), exhibited catalytic activity against guaiacol [21]. Chen et al. (2024) suggested that protein@Cu hNFs might be used as sensors for the reveal of pyridaben [22]. Koshy et al. (2024) declared that hNFs obtained by using protein (silk sericin) could be applied in ciprofloxacin (antibiotic) degradation [23]. According to another study, it exhibited catalytic activity against the neutral red dye of hNFs obtained via the coordination of DNA and the Cu+2 [24].

The synthesis of hNFs occurs by the coordination of amide, hydroxyl, or carboxyl groups of organic components and bivalent elements. The synthesis of hNFs by using plant and organic wastes containing these biomolecules instead of DNA, enzymes, and amino acids attracts attention because they are cheap and easy to synthesize [25, 26]. Anticancer activity of hNFs synthesized via coordination of Tribulus terrestris and bivalent ions (Cu (II), Zn (II), and Co (II)) against lung cancer (A549) cells was recorded [27]. Antimicrobial, antioxidant, and catalytic properties of Cu-based hNFs synthesized through Saffron (Crocus sativus), Parazoanthus axinellae (anemone), Umbilicaria decussata (lichen) and Aspergillus terreus (fungal) extracts are the trend studies of recent years [28,29,30,31]. According to a study by Altınkaynak et al. (2018), egg white and Cu coordinated hNFs exhibited peroxidase-like activity and decolorized Direct Blue dye by 76% [32].

In this study, the Elaphe sauromates, a non-poisonous snake species [33], was selected as an organic component to coordinate with Cu+2. In a previous study, it was reported that shed of E. sauromates contains α-keratin, fragile β-keratin, and many proteins and peptides [34]. The proteins found in the shed snakeskin (SSS) used in this study can coordinate with Cu+2 with the amine groups included, making SSS a good candidate for hNF synthesis. Herein, we report for the first time the synthesis of hNFs at medium pH ranging from 5 to 9 by coordination with SSS (different concentration) and Cu(II). SSS was selected for the synthesis of hNFs due to the presence of amine-group containing proteins, which reduces hNF production. Furthermore, antioxidant, antimicrobial, anticancer, anionic, and cationic dye degradation and guaiacol oxidation properties of Cu@SSS hNFs were determinded.

Materials and methods

Synthesis of Cu@SSS hNFs

Ventral portions of SSS were obtained from Elaphe sauromates (Fig. 1) which were supplied by the Anatolian Wonderland Zoo, Kayseri, Türkiye Dr. Coşkun TEZ identified the shed snakeskins at Erciyes University Department of Zoology. CuSO4.5H2O and SSS extract (10g/100ml dH2O) at different concentrations (0.5 and 1 ml) were added into PBS buffer with different pHs (pH 5–9) [25]. Vortexed (for 1 min) solutions were held at room temperature for 3 days in a dark medium and were characterized after centrifugation (10.000 rpm for 20 min) and drying (70 °C).

Fig. 1
figure 1

Images of SSS of Elaphe sauromates

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Characterization of Cu@SSS hNFs

Morphologies, inorganic and organic components, and crystal structures of Cu@SSS hNFs were determined. FE-SEM (Zeiss Gemini 500) images were used to determine the morphologies of hNFs. The presence of inorganic components in their structures was determined by EDX (EDX- ZEISS GEMINI 500) analysis. The crystallinity of Cu@SSS hNFs was examined by using an XRD (Panalytical, Empyrean) diagram. The presence of organic components in their structure was revealed by FT-IR (PERKIN ELMER, 400) peaks.

Antioxidant activity of Cu@SSS hNFs

The antioxidant activity of Cu@SSS hNFs was detailed by determining absorbance changes due to oxidation of the model substrate DPPH (2,2-diphenyl-1picrylhydrazyl). Based on the literature [35], after adding increasing concentrations from 0.165 mg/ml to 10 mg/ml Cu@SSS hNFs to the DPPH solution (0.1 mM), the absorbance of the incubated (for 30 min, in dark room) solutions was read on the spectrophotometer at 517 nm. Blank readings were performed using the same volume of ethanol (99.5%) instead of the DPPH solution. Antioxidant activities of hNFs were calculated with the formula below.

$$\text{Scavenging activity }({\%}) = [({\text{Abs}}_{\text{C}}-( {\text{Abs}}_{\text{S}}- {\text{Abs}}_{\text{B}})]/ {\text{Abs}}_{\text{C}} \times 100$$

Absc = control: distilled water, AbsS: sample, Absb = blank: ethanol (99.5%).

Guaiacol degradation ability of Cu@SSS hNFs

In order to observe the ability of Cu@SSS hNFs to oxidize guaiacol via its catalytic pathway, 1 ml each of H2O2 (22.5 mM), guaiacol (45 mM), 3 × 10–3 g Cu@SSS hNFs were transferred to test tubes containing PBS (1 ml, pH 6.8) and vortexed for 1 min, then centrifuged at 4000 rpm [26]. Absorbance changes against the blank solution (without Cu hNFs) were recorded with spectrophotometer readings at 470 nm.

Dye degradation property of Cu@SSS hNFs

Previous literature was applied to examine the capacity of Cu@SSS hNFs to degrade methylene blue (cationic) and brilliant blue (anionic) at different pHs [25]. For this purpose, 1 ml of dye was transferred to 10 mM, 50 ml PBS buffer at increasing pH (5, 7.4, and 9) containing H2O2 prepared and vortexed. The absorbance changes for methylene blue observed at 664 nm by spectrophotometric measurements of the solutions incubated in the dark were used in the formula below to determine the percentage of dye removal ability of NFs. Spectrophotometric readings were taken at 590 nm to determine degradation of brilliant dye.

$$[\text{Dye degradation }({\%}) = [({\text{Abs}}_{0}-{\text{Abs}}_{1})\text{ x }100/ {\text{Abs}}_{0}]$$

Abs0 first absorbance value, Abs1 last absorbance value.

Antimicrobial property of Cu@SSS hNFs

Gram + (Staphylococcus aureus) and Gram—(Pseudomonas aeruginosa, Escherichia coli) pathogenic bacteria were used as model microorganisms in this test. The method involving the microdilution technique was applied to evaluate the MIC values of Cu@SSS hNFs [26]. Two-fold dilutions of Cu@SSS hNFs (concentration from 4 µg/ml to 512 µg/ml) were transferred to Muller Hinton broth with 1 × 108 CFU/ml. After the tubes were kept at 37 °C (1 day), the MIC value was determined by considering the turbidity in the tubes. Negative and positive control tubes were adjusted without bacteria and only bacteria tubes, respectively. The test was triplicated.

Anticancer activity of Cu@SSS hNFs

MTT protocol was used to determine the cytotoxicity of Cu@SSS hNFs. Human lung carcinoma cell lines (A549 (ATCC)) were incubated at 37 °C (5% CO2,) in DMEM (Dulbecco's Modified Eagle's Medium)/F12 K (containing 10% fetal bovine serum with 1% penicillin–streptomycin). Cu@SSS hNFs dissolved in distilled water were serially diluted (from 250 µg/ml to 1.9 µg/ml) and transferred to the plates containing the cell line (density:12.500 cells/well) for incubation (overnight). After the 96 well plates were incubated for 24 h, absorbance readings were taken with a spectrophotometer at 572 nm [27].

Results

Characterization of Cu@SSS hNFs

FE-SEM images of Cu@SSS hNFs formed in PBS (pH = 7.4) by using 0.5 and 1 ml extract as the organic component are given in Fig. 2a and b. In the presence of SSS at low concentration (0.5 ml), it was determined that the diameter of hNF was 12.81 µm, and the petals were loosely arranged and had a structure far from a full flower morphology. It was noted that hNF synthesized by increasing the concentration of SSS extract (1 ml) had a diameter of approximately 3 µm. Optimum synthesis of hNFs was achieved by coordination of 1 ml SSS extract and Cu+2 at pH 7.4 medium. The diameters of Cu@SSS hNFs obtained by using 0.5 and 1 ml of extract in alkaline PBS buffer were measured at 11 µm and 6 µm, respectively (Fig. 2c and d).

Fig. 2
figure 2

FE-SEM images of Cu@SSS hNFs. a diameter of Cu@SSS hNFs synthesized at pH 7.4 with 0.5 ml SSS extract, b diameter of Cu@SSS hNFs syntheszied at pH 7.4, by using 1 ml SSS extract, c diameter of Cu@SSS hNFs synthesized at pH 9 by using 0.5 ml SSS extract, d diameter of Cu@SSS hNFs synthesized at pH 9 by using 1 ml SSS extract

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It was noted that the petals of hNFs synthesized with low concentration extract in alkaline medium had a loosely arranged structure, while at high concentration there were deteriorations in the petal and pore structures. hNFs synthesized under optimum conditions (PBS pH of 7.4, 1 ml extract) were used in characterization and other tests. Elemental composition of hNFs was revealed through the EDX diagram (Fig. 3). Through XRD analysis (Fig. 4), the peaks observed at 2θ = 9°, 13°, 18°, 20°, 24°, 30°, 34°, 37°, 41.52°, 45°, 54°, and 56°. Through FT-IR analysis, absorbances were recorded at 2926, 1621, 146, 1034, 986, 624, and 558 cm−1 (Fig. 5).

Fig. 3
figure 3

EDX analysis of Cu@SSS hNFs

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Fig. 4
figure 4

XRD diagram of Cu@SSS hNFs. Black line indicates with JCPDS Card 00–022 –0548, red line indicates synthesized hNF

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Fig. 5
figure 5

FT-IR spectra of Cu@SSS hNFs

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Antioxidant activity of Cu@SSS hNFs

The free radical (DPPH) scavenging efficiency of Cu@SSS hNFs with optimum morphology was examined spectroscopically. It has been detected that Cu@SSS hNFs exhibited DPPH scavenging ability (R2 = 0.5612) depending on the concentration increase (Fig. 6). Our results show that the 2.07 mg/ml of Cu hNFs were required to scavenge 50% of DPPH.

Fig. 6
figure 6

Antioxidant activity of Cu@SSS hNFs

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Dye degradation activity of Cu@SSS hNFs

It was determined that hNFs (synthesized at pH 7.4, by using 1 ml extract) synthesized with SSS extract removed methylene blue, a cationic dye, at the statistically highest level at pH 9 (75%) and the lowest at pH 5 (44%) (Fig. 7a). Additionally, an increase in the degradation of methylene blue dye was observed with increasing time (Fig. 7b). The statistically highest degradation (68%) of brilliant blue was recorded in acidic (pH 5) PBS buffer (Fig. 8).

Fig. 7
figure 7

Dye degradation activity of Cu@SSS hNFs against methylene blue. a degradation%, b time-dependent absorbance change

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Fig. 8
figure 8

Dye degradation activity of Cu@SSS hNFs against brilliant blue. a degradation%, b time-dependent absorbance change

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Oxidation of guaiacol by Cu@SSS hNFs

The ability of hNFs to degrade guaiocol through oxidation was recorded by spectrophotometric measurements. Depending on the exposure time, hNFs were determined to degrade guaiacol (Fig. 9). It was determined that the absorbance of guaiacol increased approximately 3 times from 0.075 nm to 0.235 nm.

Fig. 9
figure 9

The catalytic activity of Cu@SSS hNFs to guaiacol

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Antimicrobial activity of Cu@SSS hNFs

In this study, the antimicrobial properties of Cu@SSS hNFs with optimum morphology were investigated against three Gram (-) and Gram ( +) strains. The bacterial growth inhibitory activities caused by Cu@SSS hNFs were discussed by determining the MIC. The minimum inhibition concentrations of hNFs synthesized with SSS extract against P. aeruginosa and E.coli bacterial strains were identified at 30 µg/ml. For the S. aureus strain, the MIC was determined at 60 µg/ml.

Anticancer activity of Cu@SSS hNFs

It was determined that exposure of A549 cells to Cu@SSS hNF caused loss of viability in the cell line (Fig. 10). Increasing concentrations of Cu@SSS hNFs (1.9–250 µg/ml) were applied to A549 cell lines, and a statistically significant decrease in cell viability was observed at 15.6 µg/ml concentration application. In this experiment, the IC50 of Cu@SSS hNFs against A549 cell lines was calculated as 56.4 µg/ml.

Fig. 10
figure 10

Anticancer activity of Cu@SSS hNFs

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Statistical analysis

Data were evaluated using the IBM SPSS Statistics Standard Concurrent User V 29 (IBM Corp., Armonk, New York, USA) statistical package program. Descriptive statistics were given as mean ± standard deviation. The normal distribution of data belonging to numerical variables was evaluated using the Shapiro Wilk normality test. The homogeneity of variance of the groups was analyzed using the Levene test. The level of deterioration according to the groups was compared using one-way analysis of variance. The Duncan test was used as a multiple comparison test. The p<0.05 value was considered statistically significant.

Discussion

Characterization of Cu@SSS hNFs

Details of the synthesis mechanism of hNFs in PBS buffer have been explained in previous studies [21, 27] and the introduction part of of this article. In the nucleation phase, where the synthesis begins, metal-phosphate crystals (Cu3(PO4)2) are produced by the coordination of the phosphate groups (PO43−) originating from the PBS buffer and the Cu+2 (bivalent metal) inorganic component. The metal-phosphate crystals formed bind to the reactive groups (amine, diol) of the organic component. During the growth phase, the coordination of metal-phosphate crystals and SSS extract (organic ingredient) continues, petals forming hNFs are formed, and these petals are connected to each other by the organic component acting as a glue. As hierarchical growth reaches the saturation phase, hNF synthesis is completed. In the three-step synthesis process of hNFs, the pH of the PBS where the synthesis takes place and the concentration of inorganic and organic ingredients are critical factors in this coordination reactionas they affect the synthesis of hNFs and the formation of their morphology. In the acidic PBS buffer, the organic component-Cu3(PO4)2 coordination weakens due to the repulsion force between the protonated organic component and Cu+2. This coordination reaction occurs through the nucleation zone of the organic component. The low concentration of organic components provides a low concentration of nucleation sites in the reaction medium and causes the synthesis of hNFs with different morphologies [23, 36]. With the presence of high concentrations of organic components, it is thought that (i) metal-extract complex structures are formed and the growth phase of hNFs is prevented [37], (ii) excess extract not involved in the coordination reaction blocks the pores of hNFs and disrupts their morphology [38]. However, it has been reported that when organic components are not used in the synthesis, only primary phosphate crystals are formed and no hNFs are formed as a result of the coordination of Cu+2 with phosphate from PBS buffer [39, 40]. In previous studies, consistent with our studies, it has been declared that acidity/alkalinity of medium, and concentration of organic components have notable effects on successful synthesis and morphological characteristics of hNFs.

The existence of Cu and other elemental components (O, C, N, and P) in the content of Cu hNFs synthesized through SSS as an organic component was shown by EDX analysis. The weight percentage of O, C, N, P, and Cu in the flower-shaped hybrid nanostructures are given as 30.57, 47.95, 3.55, 3.76, and 6.94, respectively. In addition, the atomic percentage values of flower-shaped hybrid nanostructures were determined as 28.62, 59.8, 3.79, 1.82, and 1.64, respectively. By XRD analysis, 2θ = 9°, 13°, 18°, 20°, 24°, 30°, 34°, 37°, 41.52°, 45°, 54°, and 56° correspond to Cu3(PO4)2 and consist with JCPDS Card 00–022 –0548 [20, 25, 26]. Structural organic components of hNFs were revealed by using FT-IR diagrams. Amine groups (N–H) were associated with the peaks observed at wavelengths of 2926 and 1146 cm−1. The entity of aromatic bond (C = C) was signed by absorbance observed at 1621 cm−1. Other peaks (1034–558 cm−1) observed in the FT-IR diagram match phosphate groups (P–O, P = O, PO4−3). However, data obtained from FT-IR analysis of SSS extract indicate that SSS extract contains amine and diol groups and therefore can coordinate with Cu+2. In our study, the presence of Cu and amine in EDX and FT-IR analyses of hNFs is compatible with the synthesis mechanism of hNFs.

Antioxidant activity of Cu@SSS hNFs

Antioxidants have been proven to be helpful therapeutic and preventive agents and try to minimize this damage by dealing with oxidative impairment caused by reactive oxygen or free radical species [41]. It has been reported that antioxidants reduce and clean DPPH as a result of their reaction with DPPH by releasing hydrogen [42]. According to this study, hNFs synthesized by hybridization of SSS and Cu+2 have concentration-dependent DPPH scavenging activity. It has been determined that the scavenging activity of safranal-based hNFs against DPPH depends on the medium pH and concentration [28]. The IC50 value of Oxalis corniculata leaf extract-based Zn NFs against DPPH was determined at 14.72 mg/ml [43]. Kiani et al. (2024) reported that the DPPH scavenging activity of Ag-doped ZnO NPs synthesized with Lavandula stoechas extract increased with the concentration increase from 250 μg/ml to 2000 μg/ml [9]. The DPPH scavenging activity of AgNPs synthesized with Petroselinum crispum extract increased from 47 to 96% with the concentration increase of Ag NPs from 31.25 μg/ml to 250 μg/ml [10]. Consistent with our data, previous studies have declared that the antioxidant activities of nanomaterials depend on concentration, consistent with our data [35, 44, 45].

Catalytic activity of Cu@SSS hNFs: degradation of methylene blue, brilliant blue, and guaiacol

According to our catalytic activity findings, SSS coordinated Cu hNF degraded methylene blue in the highest alkaline and lowest acidic medium. It has been noted that previously synthesized CuO-ZnO nanocomposites degrade methylene blue dye the lowest in an acidic medium and the highest in an alkaline medium [46]. Similar to our results, ZnO nanoparticles synthesized with curcumin extract adsorbed three different cationic dyes in the highest alkaline medium [47]. In Khan and co-workers’ (2024) study, it was revealed that the crystal violet dye was highly degraded by citrate silver nanoparticles in a basic reaction solution [48]. It has been documented that under alkaline conditions, negatively charged hNFs might attract cationic dyes more effectively [46,47,48]. Our findings from the catalytic study are compatible with the literature showing that hNFs degrade methylene blue at a high rate in an alkaline medium. The weak catalytic activity exhibited by hNFs in the acidic medium in our study is explained by the repulsion force occurring between the protonation of hNFs and the cationic dye. The highest catalytic degradation of brilliant blue by hNFs in an acidic medium might be explained by the fact that positively charged hNF attracts the negatively charged anionic brilliant blue. Additionally, in our catalytic test, measurements of absorbance changes occurring with the oxidation of guaiacol were recorded. It is thought that hNFs cause the oxidation of guaiacol and transform it into 3,3-dimethoxy-4,4-diphenoquinone [49]. Consistent with relevant literature, we determined that Cu@SSS hNFs can oxidize guaiacol with peroxidase-like activity [26, 28, 30, 35].

In previous studies, the enhanced activities exhibited by hNFs were associated with their surface width, porosity, and 3D morphology [49, 50]. The catalytic performance of hNFs against substrates such as guaiacol and dyes has been mentioned via the Fenton oxidation process [21, 23, 30].

$${\text{Cu}}^{2+} + {\text{H}}_{2}{\text{O}}_{2}\to {\text{Cu}}^{+} + {\text{HOO}}^{0} + {\text{H}}^{+}$$
(1)
$$\text{Cu}^+ + {\text{H}}_{2}{\text{O}}_{2}\to {\text{Cu}}^{+2} + {\text{HO}}^{0} + {\text{H}}^{+}$$
(2)

Free radicals generated due to the reaction between Cu and H2O2 cause catalytic degradation by breaking down the model substrate.

Antimicrobial activity of Cu@SSS hNFs

We declare that hNFs exhibit effective antibacterial activity towards Gram ( +) and Gram (-) strains with MIC values of 60 and 30 µg/ml. The MIC values ​​of Lavandula stoechas extract-based Ag@ZnO NPs against E. coli and S. aureus strains were determined as 62.5 and 250 µg/ml, respectively [9]. MIC values ​​of Petroselinum crispum extract-based Ag NPs against E. coli and S. aureus strains were measured as 31.25 and 125 µg/ml, respectively [10]. Hashemi et al., (2023a) reported that Ag NPs synthesized with different plant extracts exhibited different levels of antimicrobial activity [2]. The MIC value of Axinyssa digitata and Cu+2 directed synthesized hNFs was 32 μg/ml against examined two strains [51]. Demirbaş et al., (2023b) calculated the MIC of Usnea antarctica-coordinated Cu-based hNFs towards E.coli at 125 μg/ml, while it was determined at 62.5 μg/ml against S. aureus μg/ml [30]. In another study, the antimicrobial effect of cherry stalk-based Cu hNFs against E. coli was recorded at 2.5 mg/ml [35]. Researchers have associated the antibacterial activities of hNFs with the Fenton mechanism. Free radicals formed after the reaction cause oxidative damage to the bacterial cell wall and provide antimicrobial properties [52].

Anticancer activity of Cu@SSS hNFs

According to the data we obtained from the study, Cu@SSS hNFs exhibit anticancer activity against the A549 cell line. Somtürk Yılmaz et al., (2024) examined the anticancer activities of Co, Zn, and Cu hNFs synthesized via Tribulus terrestris against lung cancer (A549) cell lines [27]. Researchers reported that Co and Cu hNFs synthesized with T. terrestris extract coordination showed the highest and lowest anticancer activity against A549 cell lines, respectively. In a study evaluating the toxicity of hNFs, it was emphasized that the Persea americana-directed Cu hNFs had more cytotoxicity to mouse fibroblast cells compared to the Zn hNFs synthesized with the same extract [53]. International Standard Organization (ISO 10993–5) considers a decrease in cell viability of more than 30% within the framework of cytotoxicity. According to our data, a statistically significant decrease in A549 viability was detected at 15.6 µg/ml exposure. Additionally, Cu hNFs reduced the cell viability below 70% at 31.25 and higher concentrations. Gwon et al., (2021) reported that Si@NiOOH nanoflowers had low cytotoxicity against mouse embryonic fibroblasts [54]. Shinde et al., (2020) determined that TiO2 nanoflowers have time-dependent cytotoxicity against HepG2 cell lines [55]. The toxicity caused by hNFs might be attributed to the fact that Cu+2 released from the structure of hNFs causes oxidative damage. If the cellular defense mechanism, consisting of enzymatic and non-enzymatic defense systems, cannot cope with oxidative stress, biomolecules are damaged and apoptosis occurs [55].

Conclusion

In the present study, SSS, an organic waste, was used to obtain hNFs by hybridization of organic and inorganic inputs. Using biological waste instead of enzymes and amino acids as organic components provides a cost advantage. The coordination ability of Cu+2 with the amine groups contained in the proteins found in snakeskin enabled the synthesis of hNFs. The pH of the PBS buffer affects the interaction of the primary phosphate crystals with the organic component by affecting the molecular charge of the organic components. Therefore, the synthesis of hNFs was synthesized depending on the pH of the medium. It has been noted that Cu@SSS hNFs display concentration-dependent antioxidant properties. In addition, the catalytic and antimicrobial activities exhibited by hNFs have been associated with the Fenton mechanism. According to this mechanism, free radicals formed after the reaction caused the degradation of the substrate. The cytotoxic effect of hNFs against the A549 cell line has been demonstrated. It is thought that hNFs synthesized with SSS extract can be used in anticancer, antioxidant, antimicrobial, and environmental technologies.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We would like to thank the Proofreading & Editing Office of the Dean for Research at Erciyes University for copyediting and proofreading service for this manuscript.

Funding

Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).

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Authors and Affiliations

  1. Department of History of Veterinary Medicine and Deontology, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Türkiye

    Cagri Caglar Sinmez

  2. Department of Aquatic Animals and Diseases, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Türkiye

    Fatih Doğan Koca

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  1. Cagri Caglar Sinmez
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Contributions

Cagri Caglar Sinmez: Planning of the study, Identification of snake, antioxidant and catalytic activities of nanoflowers. Fatih Doğan KOCA: Planning of the study, Methodology, Synthesis and characterization of Nanoflower, Writing.

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Correspondence to Cagri Caglar Sinmez.

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Sinmez, C.C., Koca, F.D. Synthesizing hybrid copper phosphate (Cu3(PO4)2) nanoflowers using Cu+2 and shed snakeskin: antioxidant, antibacterial, anticancer, guaiacol, anionic, and cationic dye degradation properties. J Biol Eng 19, 1 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13036-024-00464-x

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Journal of Biological Engineering

ISSN: 1754-1611