Biosynthesis of Fe₃O₄ Nanoparticles Using Egg Albumin: Antifungal, Dielectric Analysis and Photocatalytic Activity

Abstract

The use of chemical pesticides has led to adverse effects on human health and the environment, prompting the exploration of alternative solutions. This study successfully biosynthesized iron oxide nanoparticles (Fe₃O₄ NPs) using chicken egg albumin, which served as reducing and capping agents, and evaluated their antifungal efficacy against Macrophomina phaseolina. The fungicidal potential of Fe₃O₄ NPs was assessed in vitro, demonstrating enhanced inhibition of M. phaseolina’s radial growth with increasing concentrations from 100 ppm to 300 ppm. Dielectric properties were also studied, revealing advantageous current conduction processes and conductive network development with temperature variation, which is particularly beneficial in the low-frequency range. At a fixed pH, dielectric studies showed increased mobile carrier movement and polarization with rising temperature at a fixed frequency. The photocatalytic activity of Fe₃O₄ NPs was assessed for the degradation of methylene blue (MB), an organic dye, under solar irradiation. In this study, Fe₃O₄ NPs photocatalysts achieved 89% (MB) degradation within 75 min. This research underscores the potential of using chicken egg albumin for the biosynthesis of Fe₃O₄ NPs. It offers a promising alternative for plant disease control and highlights their suitability for integration into eco-friendly plant protection strategies.

1. Introduction

Fast population growth and the effects of climate change provide serious problems for agriculture. Yield losses brought on by various plant diseases and pests are another factor contributing to the difficulty in meeting food demands. Fungi, which cause more than 25% of plant illnesses, are one of the main factors limiting yield [1]. Nanotechnology, which involves the manipulation of matter at the atomic and molecular levels within the nanoscale range (typically 1–100 nm), has brought transformative advancements across multiple domains, including electronics, healthcare, energy, and environmental science [2,3,4,5,6]. At the heart of nanotechnology lies nanomaterials, characterized by their distinctive physical, chemical, and mechanical properties that differ from bulk materials due to their enhanced surface-to-volume ratio, quantum effects, and precisely engineered nanostructures [7,8,9,10,11].

To fully harness the potential of nanotechnology in plant disease protection and management, it is imperative to investigate the effects and effectiveness of nanosized particles on microorganisms and their usage in the synthesis of fungicides and pesticides [12,13]. Nanotechnology has gradually benefited the construction of antimicrobial materials to control harmful organisms that impact people, animals, and crops [14]. The challenges created by growing environmental stressors and food needs in agriculture have created a viable solution. It provides a range of strategies to raise crop yields, such as applying nano pesticides to keep plants safe from diseases and nano fertilizers to increase soil quality [15,16]. By encapsulating substances at the nanoscale, nanomaterials can release molecules gradually, minimizing environmental impact and lowering the amount needed. Macrophomina phaseolina (Tassi) is a most destructive fungal plant pathogen [17]. It is distributed globally and is particularly problematic in arid climates, infecting over 500 plant species worldwide [18]. The root-rot fungus is an emerging disease of green gram caused by M. phaseolina and is responsible for significant losses in production [19].

In addition to being a sustainable and biocompatible capping and reducing agent, egg albumin’s biochemical diversity—which includes carboxyl, hydroxyl, and amino functional groups—is essential for stabilizing nanoparticles. Strong coordination with metal ions is made possible by these functional groups, which direct the development and nucleation procedure while inhibiting aggregation [20,21]. Because of this, Fe₃O₄ nanoparticles produced using egg albumin usually have a homogeneous size distribution, good colloidal stability, and improved biological compatibility, which makes this method especially advantageous for the production of environmentally benign nanomaterials.

According to Bachheti et al. [22], Fe₃O₄ NPs are a safe and effective antifungal agent with many uses in the medical, agricultural, and environmental fields. According to Parveen et al. [23], Fe₃O₄ NPs produced in an environmentally friendly manner with tannic acid demonstrated antimycotic action against Aspergillus niger, Cladosporium herbarium, Alternari aalternata, Penicillium chrysogenum, and Trichothecium roseum. Euphorbia helioscopia leaf extract biogenic Fe₃O₄ NPs were spherical, with a particle size range of 7–10 nm, and they were effective against the C. herbarium strain of fungal infection [24]. This work highlights several significant benefits over alternative synthesis techniques and capping agents, indicating the potential of employing egg albumin for environmentally friendly Fe₃O₄ NPs synthesis. As a reducing and capping agent, egg albumin provides a biocompatible, affordable, and sustainable substitute. Even though co-precipitation is a well-established chemical synthesis method with magnetic solid characteristics, it frequently uses toxic chemicals, negatively affecting the environment and reducing biocompatibility [25]. Egg albumin-synthesized Fe₃O₄ NPs are superior in terms of biocompatibility, stability in biological media, and environmental impact. These qualities combine to make egg albumin a viable option for biosynthesis, adhering to environmentally conscious ideals while preserving affordability and usefulness in real-world uses. Chinnadurai et al. [26] present a semi-green method for fabricating Fe₃O₄ NPs utilizing Channa straitus as part of a green strategy. They also claim that the synthesis of Fe₃O₄ NPs consists of three steps: initiation, development, and termination in mucus-stabilized NPs.

The optical properties of the prepared Fe₃O₄ NPs significantly influence their biological activities, particularly in applications such as imaging, photothermal therapy, and drug delivery. High optical transmittance and specific absorption characteristics enable these NPs to interact effectively with light, which can be harnessed for therapeutic and diagnostic purposes. Moreover, the surface plasmon resonance (SPR) effect in NPs can enhance cellular uptake and bioavailability, thereby improving the efficacy of drug delivery systems. Overall, the optical properties of Fe₃O₄ NPs play a crucial role in their interaction with biological tissues and cells, enhancing their functionality and effectiveness in various biomedical applications. Despite the promising potential of synthesizing Fe₃O₄ NPs using egg albumin, several research gaps remain. The optimization of synthesis parameters, such as the concentration of albumin, reaction temperature, pH, and time, needs thorough investigation to achieve consistent and uniform NPs production. Additionally, the exact mechanisms by which egg albumin facilitates the reduction and capping processes at the molecular level are not fully understood and require detailed study. The long-term stability of these NPs under various environmental conditions is another area that lacks comprehensive data. Finally, the scalability and economic feasibility of this biosynthesis method for large-scale production have yet to be evaluated, along with its environmental impact and potential for commercialization. Addressing these gaps will enhance the applicability and sustainability of this biosynthesis approach. The innovative synthesis of Fe₃O₄ NPs using chicken egg albumin represents a significant advancement in green nanotechnology. The albumin reduces iron salts to Fe₃O₄ NPs while simultaneously capping the NPs, preventing agglomeration and ensuring uniform size distribution. Furthermore, using albumin contributes to the sustainability of the synthesis process by utilizing a natural, biodegradable material. Water quality, marine life, and the food chain are all negatively impacted by organic contaminants. The textile industry uses a lot of organic chemicals and dyes, which are emitted as effluent throughout the dyeing and finishing operations. Over time, these organic dyes contaminate water sources due to their low biodegradability. This contamination poses a threat to ecosystems and human health [27]. However, lately, promising nanomaterials with high surface area, including Fe₃O₄, ZnO, and TiO₂, have been enhanced and achieved effectiveness in the photodegradation of organic contaminants at the lab level. Additionally, ZnO NPs are used as a nanocatalyst to remove 2-Chlorophenol and on the degradation of lignin [28], TiO₂ is used on the photocatalytic degradation of tebuconazole (TEB), Congo red, and cefotaxime [29,30], and Fe₃O₄ NPs and doped Fe₃O₄ are used on the photocatalytic degradation of carbol fuchsin dye [31], furacilin [32], and doxorubicin hydrochloride drugs [33]. Egg albumin provides a biocompatible, economical, and sustainable substitute for conventional co-precipitation, sol-gel, or hydrothermal processes, which frequently call for hazardous chemicals (such as ammonium hydroxide, surfactants, or stabilizers), high temperatures, and intricate reaction setups. Albumin is a naturally occurring protein-based bio template and stabilizing agent that inhibits aggregation, improves particle dispersion, and regulates shape without producing harmful by-products. Compared to alternative green synthesis methods employing plant extracts, microbes, or polysaccharides, albumin delivers constant functionalization due to its well-defined amino acid composition and intrinsic capacity to bind metal ions. Additionally, albumin-derived Fe₃O₄ NPs frequently display increased magnetic characteristics and stability, making them attractive for medicinal and environmental applications. The manuscript’s claim of originality and excellence would be reinforced with a thorough discussion that includes comparable data on particle size, crystallinity, magnetic characteristics, and efficiency in target applications. This innovative approach underscores the potential of utilizing natural biomolecules in nanomaterial synthesis, paving the way for eco-friendly and sustainable advancements in various applications, including biomedicine and agriculture. This work explores the possibility of using albumin extract as a sustainable and environmentally friendly way to synthesize antifungal Fe₃O₄ NPs. As a result, it is simple to utilize and obtain as a reducing agent in the NPs making process. This framework utilized chicken egg albumin for the biosynthesis of Fe₃O₄ NPs, serving as a substitute for harmful reducing or capping chemicals. We analyzed synthesized Fe₃O₄ NPs using XRD, FTIR, SEM, TEM, and UV-Vis NIR spectrophotometry, assessed their antifungal efficacy against M. phaseolina, and conducted dielectric analysis. The degrading potential of the synthesized Fe₃O₄ NPs for the removal of MB dye from water in the presence of sunlight was also evaluated.

2. Results and Discussion

2.1. X-Ray Diffraction (XRD)

Using the Scherrer formula as in Equation (1), the average crystalline size (D) of Fe₃O₄ NPs was determined from the XRD pattern [34].

$$D={\displaystyle \frac{\mathrm{k}\mathsf{\lambda }}{\mathsf{\beta }\mathrm{c}\mathrm{o}\mathrm{s}\mathsf{\theta }}}$$

(1)

The wavelength (CuK_α) is utilized, where k represents the shape factor (0.9), θ is the diffraction angle, and β is the full width at half maximum (FWHM). The sample’s phase structure was determined using XRD. The XRD pattern was detected at 30.16°, 35.50°, 43.17°, 53.50°, 57.29°,62.80°,71.39°, and 74.28° corresponding to cubic Fe₃O₄ planes (220), (311), (400), (422), (511), (440), (620), and (533), respectively, were compared with JCPDS file 65–3107, and no impurity phase were detected, as shown in Figure 1a. The crystallite size of Fe₃O₄ NPs corresponding to the maximum intensity peak was determined using the (311) plane. The crystallite size was calculated using the Scherrer formula, yielding a value of 20 nm. The structural parameters, such as lattice constant (a): 8.395 Å, lattice strain (ε): 0.0023, and dislocation density (δ): 3.56 × 10¹⁵ lines/m² for the prepared Fe₃O₄ NPs were calculated using equations based on XRD data (

Table 1. Presenting experimental results for optimizing the synthesis conditions of Fe₃O₄ NPs.

Parameter	Tested Values	Particle Size (nm)	Crystallinity (XRD FWHM)	Optimum Condition
pH	6, 7, 8, 9	40.2, 28.5, 20.6, 25.3	Broad, Medium, Sharp, Broad	pH 8
Temperature (°C)	30, 50, 85, 100	35.6, 30.4, 20.6, 22.8	Broad, Medium, Sharp, Sharp	85 °C
Iron Salt Conc. (M)	0.1, 0.2, 0.3, 0.4	20.6, 50.3,35.8, 18.4	Sharp, Broad, Medium, Broad	0.1 M
Reaction Time (hrs)	1.5, 4, 8, 24	20.6, 45.2, 30.8, 22.5	Sharp, Medium, Sharp, Broad	1.5 h
Calcination Temp (°C)	300, 400, 500, 600	35.2, 28.4, 20.6, 22.3	Broad, Medium, Sharp, Sharp	500 °C
Calcination Time (hrs)	3, 4, 5, 6	32.4,20.6, 28.6, 21.8	Broad, Sharp, Medium, Sharp	4 h

). The optimal values were selected based on the smallest particle size (20.6 nm) and highest crystallinity (sharp XRD peaks). The data suggest that at pH 8, 85 °C, 0.1 M iron salt concentration, 90 min reaction time, and calcination at 500 °C for 4 h, the NPs exhibit the best properties.

2.2. Fourier Transform Infrared Spectroscopy (FTIR)

The functional group of the bio-fabricated Fe₃O₄ NPs was determined using FTIR. Figure 2b depicts the FTIR spectra of Fe₃O₄ NPs. The FTIR spectrum of Fe₃O₄ NPs corresponds with prior findings, as expected. O-H stretching vibration is responsible for the broad and strong band visible at 3390 cm⁻¹. The bands at 884 cm⁻¹, 1329 cm⁻¹, and 1571 cm⁻¹ correspond to the C-O stretching vibration, the C-H deformation vibration, and the C=C stretching vibration, respectively [35]. Compared to earlier literature on Fe₃O₄ NPs, a strong absorption band developed in the FTIR spectra of the Fe₃O₄ NPs at around 569 cm⁻¹, matching the stretching mode of Fe-O [36]. The FTIR spectra of the Fe₃O₄ NPs showed the development of a prominent absorption band at about 580 cm⁻¹, which matched the Fe-O stretching mode.

2.3. Scanning Electron Microscopy (SEM)

SEM techniques were used to investigate the microstructure and surface morphology of Fe₃O₄ NPs. Figure 2a,b show the scanned images under SEM, which show highly uniform and smooth particle dispersion with negligible agglomeration. The size of the Fe₃O₄ NPs is consistent with the TEM pictures, which show spherical particles. However, particle aggregation may be seen in the micrographs. Inter-granular gaps are another feature that can be seen, indicating that the grains are more connected. Figure 2a,b demonstrates the results, which reveal less agglomerated formations with spherical particles. The non-agglomerated SEM images showed distinct and stable particles.

2.4. Transmission Electron Microscopy (TEM)

The TEM picture of the Fe₃O₄ NPs in Figure 2c,d revealed that the nanomaterial had a particle size distribution of 20.60 nm. ImageJ software calculated particle size based on the TEM images in Figure 2c,d. The nanoscale production of Fe₃O₄ NPs was validated through TEM images. Figure 2c,d depicts a narrow distribution of Fe₃O₄ NPs, with an average particle size of approximately 20.60 nm determined using Image J software. Figure 2e reveals that the SAED pattern has concentric rings, indicating polycrystalline. Figure 2f illustrates a broader distribution of Fe₃O₄ NPs. The homogeneous distribution of Fe₃O₄ NPs was predicted to improve catalytic activity. The well-dispersed average particle size using a TEM micrograph was calculated and found to be 20.60 nm.

2.5. UV-Visible Spectroscopy

UV-visible spectroscopy is among the most effective techniques for investigating the optical properties of NPs dissolved in a solvent. In the 300–800 nm region, the optical absorption of bio-transformed Fe₃O₄ NPs was examined. The correlation between k and incident photon energy is shown using Tauc’s equation (Equations (2) and (3)) [37].

$${\mathrm{k}=\mathrm{A}(\mathrm{E}-E_g)}^n$$

(2)

$$\mathrm{f}({\mathrm{R}}_{\mathrm{\infty }})={\displaystyle \frac{k}{s}}={\mathrm{A}(\mathrm{E}-E_g)}^n$$

(3)

In the above equation, ‘n’ represents a constant that may vary based on the type of electronic transition, where ‘n’ equals 1/2 for an allowed direct transition and ‘n’ equals 2 for a permissible indirect transition.$E_g$ is the band gap, photon energy, and A is a constant that depends on the properties of the NPs. Figure 3a displays the broad absorbance spectra for Fe₃O₄ NPs, measured between 300 and 800 nm. The broad spectra were observed between 340 and 460 nm due to the presence of biological entities present in the egg albumin. Tauc’s figure was used to compute the bandgap; as shown in Figure 4b, the bandgap (E_g) is obtained by extrapolating the linear region on the x-axis. The bandgap energy of 3.01 eV for the phyto-fabricated semiconducting Fe₃O₄ NPs was determined from the Tauc plot (Figure 3b), an (αhν)² vs. energy (hν) graph that was drawn from UV–Visible absorption data of Fe₃O₄ NPs. As synthesized Fe₃O₄ NPs by chicken egg albumin’s biological components caused the broad spectra between 340 and 460 nm, confirming metal oxide NPs’ absorbance.

2.6. Dielectric Study of Fe₃O₄ NPs

Table 2. Observed values of real part dielectric permittivity (ε′), the imaginary part of dielectric constant (ε″), tangent loss (tanδ), and conductivity (σ (AC) within the temperature range of 300–573 K.

Frequency	ε′	ε″	Tanδ	σ (AC) (S·m−1)
20 Hz	145,000	600,000	4.00	0.0065
0.50 MHz	67	38	1.20	0.0024
1.0 MHz	37	14	0.80	0.0016
2.0 MHz	31	10	0.60	0.0021
5.0 MHz	25	07	0.40	0.0027

presents the examination and tabulation of conductivity [σ (AC)], the real part of dielectric permittivity (ε′), the imaginary part of dielectric permittivity (ε″), and loss tangent (tanδ) as functions of temperature (T in Kelvin) at selected frequencies of 20 Hz, 0.5 MHz, 1.0 MHz, 2.0 MHz, and 5.0 MHz. The temperature and frequency-independent approaches to the intrinsic static dielectric constant are shown in σ (AC), ε′, ε″, and tanδ below 425 K [38]. It showed better dielectric properties ranging from 550–573 K. Furthermore, when T(K) > 573 K grows; Figure 4 illustrates that no discernible dielectric relaxation peaks were found in the loss tangent (tan δ), and the actual component of the dielectric permittivity with temperature-dependent ε’ (T) in Kelvin increases in a defined step. The relaxation mechanism is believed to be hidden by ionic conduction [39]. The relaxation peak in the tan δ curve can only be seen when the electrode polarization effect is taken out of the picture. Frequency-dependent dielectric relaxation in the Fe₃O₄ NPs is caused by charge carriers building across sample regions with varying conductivities, such as conducting grains and insulating grain boundaries, at high temperatures. Maxwell–Wagner describes this dielectric relaxation’s extrinsic cause [40].

A prominent method for characterizing the charge carrier/ion dynamical dynamics is to study frequency-dependent conductivity. The relation (σ (AC) = ε₀ε’ωtan δ) was used to calculate the σ (AC) values to investigate the effects of particle size on the alternating current conductivity of Fe₃O₄ NPs [41]. Figure 4d displays the frequency-dependent conductivity σ (AC) for each Fe₃O₄ NPs in the 300 K–573 K range. Temperature-dependent AC conductivity increases because heat generates charge carriers. To provide further insight, it can be observed that the alternating current (AC) conductivity adheres to Jonscher’s power law, expressed as (σ (AC) = dc + Afⁿ)), where σ (AC) represents the total conductivity, dc is the extrapolated direct current (dc) conductivity, n signifies the temperature, and f represents the frequency. A and F stand for material-specific temperature-dependent constant and the temperature, respectively. Figure 4 displays the temperature dependence of the fabricated Fe₃O₄ NPs. As shown in Figure 4, Fe₃O₄ NPs' dielectric constants increase sharply as temperature increases in the low-frequency range, but they tend to increase first and then become stable at higher frequencies [42]. Then, with further increase, it tends to decrease after reaching relaxation. This phenomenon is attributed to varied responses of different polarization mechanisms across various frequency ranges and is also accountable for polarization relaxation [43].

The three primary polarization mechanisms of ferroelectric materials are turning-direction polarization, space charge polarization, and displacement polarization. Of them, displacement polarization has the shortest response time (~10⁻¹³ s), allowing the polarization process to develop within the frequency range being monitored fully. However, the time required for inducing turning-direction and space charge polarization is significantly longer than displacement polarization, estimated to be around 10⁻⁵ s. As a result, this relaxation polarization can only be developed at very low frequencies. In the low-frequency range, field stress or thermal motion can prompt electrons to accumulate at the grain boundaries if the resistance of the boundaries is sufficiently high, resulting in polarization. Conversely, as the frequency increases, the pile-up effect diminishes as electrons consistently alter their direction of motion, reducing polarization. The space charge polarization resulting from non-uniform dielectric structure and other flaws like porosity, grain structure, and impurities is another component affecting lower frequencies permittivity. A space charge layer is established when a few free charges persist at the boundary surface’s center, counterbalancing each other. This allows for changes to the space field, the same as improving the dielectric characteristics. At relatively high frequencies, the creation of turning-direction and space charge polarizations is limited, except for displacement polarization. As a result, the dielectric constant’s dispersion decreases, and it exhibits a frequency-independent response. Put simply, the presence of dispersion in the dielectric constant can be attributed to the polarization mechanisms operating at different frequencies. Space charge accumulation along with substantial interfacial polarization around grain boundaries, which becomes more prevalent at low frequencies, represents the cause of the noticeably elevated permittivity seen at 20 Hz. The intrinsic heterogeneity as well as defect sites for bio-synthesized FeO₄ nanoparticles additionally affect this behavior, which is frequently observed in nanostructured metal oxides [36,44,45].

2.7. Factors Affecting Biosynthesis of Fe₃O₄ NPs

Temperature, reactant concentration, reaction time, and pH are key variables affecting the biosynthesis of Fe₃O₄ NPs employing phytochemicals. Each parameter strongly determines the form, size, and general characteristics of the synthesized NPs. A detailed explanation of how several variables affect the synthesis process may be found below.

2.7.1. pH

The pH of the reaction mixture influences the ionization state of the metal ions and phytochemicals, as well as the reduction process and the stability of the NPs. Under low acidic conditions, insufficient capping leads to an inadequate reduction of iron ions and the agglomeration of NPs. Under simple conditions, enhanced reduction and stabilization result in the production of smaller, more uniform NPs. Precipitation can happen quickly at highly high pH values, producing larger particles. For the most effective reduction and stability during the synthesis of Fe₃O₄ NPs, a pH between 8 and 10 is basic [46].

2.7.2. Temperature

Temperature affects both the kinetics of the reduction reaction and the rate at which the NPs grow. Longer growth times produce larger NPs since low temperatures slow the reaction rate. High temperatures quicken the nucleation process and enhance the reaction rate, resulting in smaller NPs due to limited development. Generally, moderate temperatures (between 60 and 80 °C) control the size and homogeneity of the NPs and the reduction rate [47].

2.7.3. Concentration of Reactants

The concentration of the biological extract and the iron salt precursor dictates the availability of reactants for the reduction process and the ensuing production of NPs. High concentrations of iron salts have the potential to produce rapid nucleation and agglomeration, resulting in more widespread and less uniform NPs. On the other hand, low iron salt content may result in inadequate reduction and subpar NPs production. When determining the optimal ratio of phytochemical extract to iron salt, the plant extract should be at an equally good ratio, with typical values for iron salt being 0.1–0.5 M [48].

2.7.4. Reaction Time

The reaction time determines the length of the reduction and growth processes, which affect the size and crystallinity of the NPs. The quick reaction time could result in partial reduction and smaller, possibly unstable NPs. Reaction times that are too long can cause aggregation, even while they allow for complete reduction and development, which produces well-formed and stable NPs. Depending on the specific biological extract and other variables, two to twenty-four hours are often needed for effective reactions [49].

2.8. Photocatalytic Study of Fe₃O₄ NPs Under Solar Light

The photodegradation of MB was investigated utilizing Fe₃O₄ NPs by exposing the MB solution to sun irradiation. The reaction kinetics were analyzed to ascertain the order of the photocatalytic degradation events, revealing that the dye degradation adhered to first-order kinetics [38]. The rate constant revealed that the degradation rate of MB was 0.012 s⁻¹ using solar light irradiation. The photocatalytic degradation efficiency of the organic dyes was calculated using the following formula, as mentioned earlier.

The MB absorbance remained relatively constant, signifying the absence of degradation activity in the absence of a photocatalyst, as evidenced during photodegradation under solar irradiation in control conditions. The solution subjected to solar radiation exhibited a reduction in absorbance, as illustrated in Figure 5A, from time t = 0 min to 75 min. The photocatalytic efficacy of MB without the presence of the photocatalyst (Fe₃O₄ NPs) under natural sunlight remained constant, as illustrated in Figure 5B. The efficacy of the photocatalytic activity of biosynthesized Fe₃O₄ NPs was evaluated through the degradation of methylene blue dye in an aqueous solution under natural sunshine. Following four successive cycles, the photocatalytic degradation of MB exhibited stability at 89%, 87%, 86%, and 85%, respectively, as depicted in Figure 5C, over a 75-min exposure to natural sunshine. This study indicates that the Fe₃O₄ NPs derived from albumin extract exhibit exceptional photocatalytic capabilities. The photocatalysis performance comparison of our Fe₃O₄ with other iron oxide-based nanomaterials is presented in

Table 3. Photocatalysis performance comparison of our Fe₃O₄ with other iron oxide-based nanomaterials.

Nanomaterial	MB Photodegradation (%)	Time (min)	K (min−1)	Reference
Fe3O4	89	75	0.0133	This work
Fe3O4@C@Ru	92.70	140	0.0176	[50]
Fe2O3/graphene/CuO	94.27	40	0.0725	[51]
Fe3O4@SiO2	97	140	0.0474	[52]
rGO-Fe3O4	98.30	80	0.0464	[53]
Fe3O4@Ag@TiO2	79.90	120	0.0120	[54]
Fe3O4/MWCNT	98.40	60	0.7398	[55]
GO-LaFeO3	91.20	150	0.0137	[56]

.

2.9. In Vitro Assessment of Fe₃O₄ NPs Against M. phaseolina

The efficacy of Fe₃O₄ NPs was examined at three different concentrations, viz., 100, 200, and 300 ppm, to determine the inhibition zones against M. phaseolina using the poison food technique (

Table 4. Efficacy of Fe₃O₄ NPs on the colonization of M. phaseolinain in vitro.

Treatment	Concentrations (ppm)	Inhibition Zone (%) of M. phaseolina After Seven Days
Fe3O4 NPs	300	51.5 a
	200	34.3 b
	100	21.5 c
	Control	0 d
ANOVA	DF	3
	Sum of Squares	4238
	Mean Squares	1412.66
	F-Calculated	209.98
	Significance	0.0000

Values ^a,b,c,d followed by same letter within a column are not significantly different according to Duncan’s multiple-range test (p ≤ 0.05).

). Fe₃O₄ NPs extract was coated with stabilizing agents, viz., silica, preventing them from aggregating and settling. The Fe₃O₄ NPs showed significant ability to inhibit the fungal growth of M. phaseolina at concentrations of 200 to 300 ppm, with an inhibition zone of 34–52%, respectively, over the respective control (p ≤ 0.05; Table 4). However, M. phaseolina was found to be most sensitive to Fe₃O₄ NPs, resulting in a growth inhibition zone of 52% at a concentration of 300 ppm over the respective control (p ≤ 0.05; Table 4). The next concentration was 200 ppm, which efficiently suppressed the radial growth of M. phaseolina mycelium by 34% inhibition zones over the respective control (p ≤ 0.05; Table 4). The lowest reduction of radial growth of M. phaseolina mycelium was recorded with a concentration of 100 ppm, with 22% inhibition zones over the respective control (p ≤ 0.05; Table 4). The experiment showed that increasing the concentration of Fe₃O₄ NPs increased the percent inhibition zone, which caused greater suppression of radial growth of M. phaseolina.

Degree of freedom (DF) refers to the number of independent values that can vary in an analysis without violating constraints. The degrees of freedom depend on the number of groups and the total sample size. The antifungal activity of Fe₃O₄ NPs was found to be effective and significantly inhibited the radial growth of the M. phaseolina. Hence, Fe₃O₄ NPs can be regarded as highly effective antifungal agents against many phytopathogenic fungi [23,57]. NPs’ mediated toxicity in fungi occurs through various mechanisms, including causing cell wall damage/membrane damage, gene regulation, DNA interactions, ion release, damage to hyphae and spores, effect on biofilm formation, reactive oxygen species (ROS) impact on mitochondria, gene regulation, and protein levels and ROS generation, resulting in a cascade of damage including lipid peroxidation and mitochondrial impairment [58,59]. Exposure to NPs promotes modifications in the fungal cell wall, encompassing surface contraction, cellular aggregation, pit and pore development, and overall deformation [60,61]. Fe₃O₄ nanoparticles can inhibit fungal growth by causing DNA damage, protein denaturation, and disrupting the cell wall and membrane integrity, and also induce lipid peroxidation through the generation of reactive oxygen species, cause ribosomal disassembly, and deactivate essential enzymes [62]. However, Fe₃O₄ NPs can bind to fungal cell walls, affecting their structure and permeability [63]. The magnetic characteristics of Fe₃O₄ enable targeted dispersion and contact with fungal cells, while their nanoscale size allows for deeper penetration and enhanced surface reactivity [64]. Moreover, Fe₃O₄ nanoparticles can cause perforations in cellular structures, damage mitochondria, release cytochrome-c into the cytosol, and elevate metacaspase levels, leading to programmed cell death [65,66].

The albumin protein from egg white plays a crucial role in the biofabrication of Fe₃O₄ NPs by acting as both a reducing and capping agent. The functional groups in albumin, such as hydroxyl, amino, and carboxyl groups, facilitate the reduction of iron salts to iron ions, leading to the formation of Fe₃O₄ NPs. Additionally, albumin’s protein structure provides numerous binding sites that cap the NPs, preventing aggregation and ensuring uniform size distribution. This capping action stabilizes the NPs in colloidal solutions, enhancing their biocompatibility and making them suitable for biomedical applications. The biocompatible nature of albumin allows for further functionalization, enabling the conjugation of drugs or targeting agents to the NPs. Moreover, using albumin in the synthesis process is environmentally friendly as it eliminates the need for toxic chemicals, making the process sustainable and safe. This biosynthesis method underscores the potential of natural biomolecules in fabricating eco-friendly and functional nanomaterials for advanced applications.

Optimizing the yield of Fe₃O₄ NPs using chicken egg albumin entails methodically adjusting crucial synthesis parameters like temperature, pH, reactant concentration, and reaction duration. The final nanomaterial was obtained by calcining the nanomaterial at 500 °C. As an example, different pH levels (6, 7, 8, 9, 10), temperatures (30 °C, 50 °C, 85 °C, 100 °C), iron salt concentrations (0.1 M, 0.2 M, 0.3 M, 0.4 M), reaction times (1.5, 4, 8, 24 h), calcination temperatures (300 °C, 400 °C, 500 °C, and 600 °C), and calcination times (3, 4, 5, and 6 h) were tested to optimize these conditions. TEM, SEM, FTIR, XRD, and UV-Vis spectroscopy were among the methods used to assess the yield and characteristics of the fabricated Fe₃O₄ NPs. The best yield and NPs quality have been achieved at 0.1 M iron (II) nitrate hexahydrate, pH 8, 85 °C, 1.5 h of reaction time, and 4 h of calcination at 500 °C. Optimization can make the biosynthesis technique practical, scalable, and ecologically benign. This optimization ensures that the biosynthesis method is efficient, environmentally friendly, and scalable for practical applications.

Iron ions (Fe³⁺ or Fe²⁺) are reduced by albumin extract to zero-valent iron (Fe⁰) by electron donation, which starts the fabrication of NPs (Fe³⁺ + reducing agent → Fe⁰). The albumin extract functional groups, like amino groups, hydroxyl, and carboxyl, help to stabilize the NPs by creating a shield that stops oxidation and guarantees that the particles are evenly distributed and stable (Fe (NO₃)₂·6H₂O+ albumin extract → capped and stabilized Fe₃O₄ NPs) [67]. Ultimately, the NPs oxidize to fabricate magnetite (Fe₃O₄) in the presence of oxygen or under controlled heating (Fe⁰ + O₂→ Fe₃O₄ or 3Fe²⁺ + 4H₂O + O₂→ Fe₃O₄ + 8H⁺). By producing biocompatible Fe₃O₄ NPs without hazardous chemicals, this economical and environmentally beneficial process complies with green chemistry principles. The research on bioengineered Fe₃O₄ NPs emphasizes their noteworthy prospects in photocatalytic and biological domains because of their distinct physicochemical characteristics and eco-friendly manufacturing techniques.

According to Ammar et al. [68], fungicide activity against M. phaseolina, Rhizocotonia solani, Fusarium sporotrichioides, F. proliferatum, F. solani, and F. oxysporum was marginally increased by zinc NPs. Chitosan NPs (@0.1%) significantly reduced the development of Alternaria alternata, M. phaseolina, and R. solani by 82%, 88%, and 34%, respectively [69]. At concentrations between 0.03 and 0.15%, chitosan NPs inhibited the growth of F. verticillioides by 20–60%, reaching complete inhibition at 0.21%. Similarly, A. alternata showed a MIC of 0.24%, and M. phaseolina reached a MIC of 0.26% [70]. The findings in this study are consistent with earlier investigations that revealed the development of the biogenic Fe₃O₄ NPs from Eichhornia crassipes leaf extract was indicated by a peak in the UV spectral data at 379 nm [71]. According to Shahid et al. [72], Fe₂O₃ NPs were present in Apis mellifera honey because of a distinct and strong peak at 350 nm. According to earlier reports, the alkyl halides were identified by distinctive transmission bands found at 540.36 and 574.83 cm¹ [73]. According to FTIR studies, all of the egg albumin’s phenols, alcohols, amines, and alkanes work together to reduce, stabilize, and cap the biogenic Fe₃O₄ NPs. Additionally, recent research has shown that iron oxide-based NPs range in size from 10 nm to 100 nm [74,75]. A TEM revealed that the size variation of the iron NPs, which have a thick surface layer and are nearly spherical, ranges from 16.2 to 18.55 nm [76]. The biomolecules of Laurus nobilis leaf extract used in the biosynthesis process were identified as the source of the matrix-like structure in which the biogenic Fe3O4 NPs were seen to be embedded [47]. The average size of the biogenic Fe3O4 NPs was 46.3 nm, and they had a spherical shape [77].

The egg albumin methodology is environmentally benign and has little influence on the environment, unlike chemical synthesis methods that necessitate severe conditions and dangerous substances. Microbial synthesis is natural and friendly, but scalability is difficult due to lengthy reaction periods and intricate biological processes. Like other green and chemical synthesis processes, egg albumin’s inherent functional groups contribute to its high functionalization potential. Fe₃O₄ NPs produced with egg albumin have significant magnetic characteristics and are stable in biological conditions, making them appropriate for medical applications. The innovative aspect of this study is biosynthesis, which results in improved structural and functional features, even though Fe₃O₄ NPs made with egg albumin have been extensively reported for various applications. The synthesis is entirely dependent on temperature phenomena, and the innovative aspect of this study is biosynthesis, which results in improved structural and functional features. Superior antifungal, dielectric, and photocatalytic performance results from the exact egg albumin-mediated synthesis that produces controlled particle size, improved crystallinity, and increased surface area. Furthermore, the research presents Fe₃O₄ nanostructures that utilize synergistic effects to enhance dielectric, antifungal, and photocatalytic capabilities. For the first time, the removal of pathogens, dielectric, and photocatalytic properties of Fe₃O₄ NPs produced by egg albumin have been investigated, providing a viable and affordable path for pathogen removal and high-performance energy applications.

3. Materials and Methods

3.1. Materials

Fresh chicken eggs were used to extract 70 mL of albumin, analytically pure chemicals such as 0.1 M iron (II) nitrate hexahydrate (Fe (NO₃)₂·6H₂O) (98% pure, Fisher Scientific, Mumbai, India), which were then utilized in the synthesis to Fe₃O₄ NPs using double-distilled water (DDW). After manually separating the egg white (albumin) from the yolk, it was filtered using a double-layer muslin fabric to remove contaminants; it was utilized without additional purification. The protein, which had an estimated purity of above 90%, was employed right away to guarantee freshness and preserve functionality. Further, potato dextrose agar (PDA) (Sigma-Aldrich, St. Louis, MO, USA) was taken in 250 mL conical flasks and autoclaved at 15 kg/cm² pressure for 15–20 min at 121 °C. After autoclaving, these conical flasks were placed under a laminar airflow chamber for a few minutes to cool down and then mixed with a solution containing 2.5 mg/L of chloramphenicol (Sigma-Aldrich, USA) (½ PDA + c) [78]. The target organic pollutant, MB (C₁₆H₁₈ClN₃S. H₂O), was provided by Biochem, Germany, and had a molecular weight of 319.85 g/mol and a purity of 98%.

3.2. Biosynthesis Synthesis of Fe₃O₄ NPs

Here, we used a sol-gel-assisted biosynthesis approach to synthesize Fe₃O₄ NPs via a biological route that was eco-friendly, economical, non-toxic, and, most notably, produced the highest possible yield of the product (Figure 6). Raw eggs were used to extract albumin, which was then employed as a naturally reducing agent. Additionally, it served as a capping and stabilizing agent, preventing NPs' aggregation and assisting in maintaining their size, shape, and surface morphology. To synthesize NPs using chicken eggs, we followed previously reported work [79]. The synthesis of Fe₃O₄ NPs was performed using 0.1 M of iron (II) nitrate hexahydrate (Fe (NO₃)₂·6H₂O) (98% pure, Fisher Scientific, Mumbai, India), 70 mL fresh chicken egg albumin, and DDW. First of all, in 100 mL DDW, 0.1 M of iron (II) nitrate hexahydrate was added, and 70 mL of freshly obtained albumin was added slowly and stirred for 90 min with a gradual increase in temperature up to 85 °C, and a precipitate was obtained. The precipitate was calcined at 500 °C for 4 h. The obtained material was dried at 85 °C for 24 h in a hot air oven (SSK Enterprises, Mumbai, India).

3.3. Characterization of Fe₃O₄ NPs

The unit cell dimension and crystallinity phase were ascertained using a Rigaku Miniflex advance diffractometer (Neu-Isenburg, Germany) (Cu anode material, 15 mA current, 30 kV voltages, wavelength (Cu) 1.541838 Å). Optics: In the analysis of Fe₃O₄ NPs at the angle 2θ, beta filtering with graphite, an automated divergence slit, and a monochromator are employed. The 2θ range for Fe₃O₄ NPs spans 5.0° to 80.0° with increments of 0.04°. A scanning electron microscope investigated surface morphology and element identification (Model No. JSM 6510 LV, make: JEOL, Japan). The surface morphology, size, crystalline structure, and shape of NPs were investigated through transmission electron microscopy (TEM) and selected area electron diffraction (SAED) patterns. Fe₃O₄ NPs powder samples were suspended in ethanol before precipitating on the grid. Subsequently, the TEM (make—JEOL, Japan; model-JEM 2100) positioned the sample for scanning. Particle size distribution analysis was performed using Image J Launcher software version (1.4.3.6.7) produced in Tokyo, Japan, incorporating broken symmetry functionalities.

The spectrum of Fe₃O₄ NPs was recorded using Fourier transform infrared (FTIR) spectroscopy, covering wave numbers from 400 to 4000 cm⁻¹ (Make—Bruker, Model: Tensor 35). This analysis aimed to scrutinize the functional bio-molecules. Each sample was prepared by compressing 200 mg of KBr, a non-absorbing medium serving as the background under high pressure. A Cary 5000 (Make: Agilent, Country: Santa Clara, California, U.S.) spectrometer with 1 cm quartz cells was used to record the UV–vis spectra at room temperature in the range of 300–800 nm. Key Sight Agilent E4990A was used to analyze the sample from 20 Hz to 5 MHz in the 300 K to 573 K temperature range at selected frequencies. An X-ray diffractometer was employed to scrutinize the crystal structure and lattice properties of Fe₃O₄ NPs.

3.4. Temperature-Dependent Dielectric Studies of Fe₃O₄ NPs

Fe₃O₄ NPs are semiconductors with a broad bandgap of 3.01 eV that exclusively absorb light in the ultraviolet region. The semiconductor Fe₃O₄ NPs were made in circular pellets with a 13 mm diameter and a thickness of 2.65 mm by coating with the silver paste on both sides and sandwiching the insulating material between them. The Fe₃O₄ powder was compressed in a hydraulic press (Athena Technology, Mumbai, India) at 5 tons of pressure for two minutes to produce uniformly dense pellets [33,80]. To guarantee consistent conductivity and good electrode contact, high-purity silver paste was added to both surfaces with a fine brush to make a uniform thickness of about 20 µm. It was then dried for 30 min at 80 °C.

The sample was scanned over 20 Hz to 5 MHz in the 300 K to 573 K temperature range at different selected frequencies. Fe₃O₄ NPs are a highly effective contender for dielectrics because they are used in microwaves. Energy storage devices greatly benefit from the dielectric characteristics of metal oxide NPs. The study examined the relationship between temperature and the dielectric permittivity of real (ε′), imaginary (ε″), dissipation factor (tanδ), and conductivity σ (a.c). The complex permittivity, which is represented by Equation (4), is crucial to the study.

$$\mathsf{\epsilon }\ast \left(\mathsf{\omega }\right)={\mathsf{\epsilon }}^{\prime }\left(\mathsf{\omega }\right)-\mathrm{i}{\mathsf{\epsilon }}^{″}\left(\mathsf{\omega }\right)$$

(4)

In this case, the real part of entity ε*(ω), represented by ε′(ω), indicates the material’s ability to store energy, while the imaginary part ε″(ω) indicates the energy loss within the metal oxide NPs in the form of heat. Equation (5) provides an experimental measurement of the real component of dielectric permittivity.

$${\mathsf{\epsilon }}^{\prime }={\displaystyle \frac{C_p\mathrm{d}}{{\mathsf{\epsilon }}_0\mathrm{A}}}$$

(5)

where

A is the round pellet’s cross-sectional area;

d is its thickness;

C_p is its capacitance. The permittivity of air is represented by ε₀ = 8.854 × 10⁻¹² F/m.

3.5. Photocatalytic Study

The photodegradation of MB was investigated utilizing Fe₃O₄ NPs by exposing the MB solution to sun irradiation. The reaction kinetics were analyzed to ascertain the order of the photocatalytic degradation events, revealing that the dye degradation adhered to first-order kinetics, as expressed by the following Equation (6) [81].

$${C_t=C}_0{e}^{-kt}$$

(6)

$C_0$ is the starting dye concentration in ppm,$C_t$ is a concentration at time t in minutes, k is the rate constant in min⁻¹, and t is time, as shown in Equation (7).

$$-\mathrm{l}\mathrm{n}{\displaystyle \frac{C_t}{C_0}}=\mathrm{k}\mathrm{t}$$

(7)

The rate constant calculated using Equations (6) and (7) revealed that the degradation rate of MB was 0.012 s⁻¹ using solar light irradiation. The photocatalytic degradation efficiency of the organic dyes was calculated using the following formula, as mentioned in Equation (8):

$$Degradation(\%)=({\displaystyle \frac{A_0-A_t}{A_0}}\times 100)$$

(8)

where $A_0$ is the absorbance of the dye solution before photo-irradiation, and $A_t$ is the absorbance of solutions in suspension after photo-irradiation for a specific time t (0 min to 75 min).

3.6. In Vitro Assessment of Fe₃O₄ NPs Against Pathogenic Fungus and Growth Inhibition

The antifungal activity of Fe₃O₄ NPs at concentrations of 100, 200, and 300 ppm against the pathogenic fungus M. phaseolina was assessed using the poison food technique used by Falck [82] under in vitro conditions. The Fe₃O₄ NPs concentration was prepared in DDW to get the desired concentration of NPs after mixing the DMSO solution (Sigma-Aldrich, USA) in the PDA media in equal amounts before pouring in sterilized Petri plates. Each concentration of Fe₃O₄ NPs was added to 20–25 mL of the potato dextrose agar (PDA) medium. The control plate (without Fe₃O₄ NPs) was maintained with a PDA medium. Subsequently, approximately 20–25 mL of the warmed molten PDA medium containing each test concentration of Fe₃O₄ NPs was poured into each sterilized Petri plate under a laminar airflow chamber. The plates with and without Fe₃O₄ NPs were inoculated by placing a 5 mm mycelial disc cut with a cork borer of M. phaseolina into the center and incubating for 28 ± 2 °C in the biochemical oxygen demand (BOD) incubator (Labtronix, Noida, India). Three replicates of each concentration and control plate were maintained. The colony diameter of all cultured plates was measured seven days post-inoculation, coinciding with the control plates achieving complete mycelial development of the pathogen. The percentage of mycelial growth inhibition of the pathogen was determined using the methodology provided by Vincent (Equation (9)) [83].

$$\mathrm{Mycelial}\mathrm{growth}\mathrm{inhibition}(\%)={\displaystyle \frac{C-T}{C}}\times 100$$

(9)

where

C = radial growth of fungus in control;

T = radial growth of fungus in treatment.

3.7. Statistical Analysis

The collected data, encompassing several features under examination, was subjected to statistical analysis using R software (version 2.14.1). The evaluated factors that indicate significant differences (p = 0.05) were identified using Duncan’s Multiple Range Test (DMRT). An ANOVA was performed using OPSTAT [78].

4. Conclusions

A thorough comparison with other biosynthesis methods and well-established chemical processes highlights the uniqueness and benefits of using egg albumin for Fe₃O₄ NPs. In an aqueous medium at normal temperature, egg albumin has an acceptable reaction time, benign reaction conditions, good yield and reproducibility, and high synthesis efficiency. Fe₃O₄ NP synthesis using egg albumin is a greener alternative to chemical production. Egg albumin, an abundant and biodegradable biomaterial, reduces, caps, and improves NP biocompatibility, facilitating synthesis. Researchers could modify synthesis parameters to increase Fe₃O₄ NP yield and uniformity. These NPs could be functionalized with biomolecules for targeted water remediation, antifungal action, and medicine. The biosynthesis of Fe₃O₄ NPs via sol-gel-assisted was safe, cost-effective, and yielded the most. X-ray diffraction revealed this sample’s good crystallinity and Fe₃O₄ NPs' cubic structure. FTIR functional group estimation showed Fe-O, indicating metal oxide formation. SEM indicated low aggregation and well-distributed NPs. Dielectric studies reveal that temperature and frequency increase mobile carrier movement and polarization to a limit, and the drop in M. phaseolina radial growth was inhibited and antifungal by Fe₃O₄ NPs. The dielectric properties and promising antifungal activity against M. phaseolina make these NPs suitable for agricultural and biomedical uses. This makes Fe₃O₄ NPs effective antifungals for sustainable agriculture. The photocatalytic activity of Fe₃O₄ NPs was assessed for the degradation of methylene blue (MB) under solar irradiation. In this study, Fe₃O₄ NPs photocatalysts achieved 89% (MB) degradation within 75 min. Finally, this biosynthesis technique must be scaled and made economically viable before commercialization. These suggestions will improve sustainable and versatile nanomaterial development, furthering science and conservation.