A novel green biosynthesis approach and structural characterization of Ag–Fe bimetallic nanoparticles using the red alga Galaxaura rugosa

Taxonomic description of the used seaweed

Species identification and taxonomical classification were done by a classification expert using fresh samples on the same collection day. Algae are identified as red algae (Rhodophyta) namely; Galaxaura rugosa (J.Ellis & Solander), family Galaxauraceae (Fig. 1). The collected alga species were classified according to the previous literature²⁶.

UV-Vis analysis of the phyco-synthesized Ag–FeBNPs

The color variation of the algal extract was obvious on visual observation from yellow to yellowish brown. This transition of color is mainly attributed to the algal active compounds that act as reducing, stabilizing, and capping agents²⁷. UV-visible spectroscopy showed the maximum absorption peak of Ag–FeBNPs at 327 nm, as shown in Fig. 2. This characteristic band can be attributed to the surface plasmon response produced by the interaction between the electrons in the conduction band on the surface of the NPs and the incoming light²⁸. This observed absorption peak is consistent with previous studies on biologically synthesized Ag–Fe bimetallic nanoparticles, where surface plasmon resonance bands were reported between 320 and 340 nm, depending on the synthesis conditions and capping agents used^29,30.

To further evaluate the optical behavior of the synthesized Ag–FeBNPs, the optical band gap energy (Eg) was estimated using the Tauc plot method. The Tauc plot derived from UV–Vis data (plotting (αhν)² vs. photon energy hν) revealed an estimated band gap of approximately 2.85 eV, indicating the semiconducting nature of the nanoparticles. This band gap value suggests potential applicability in photocatalytic and optoelectronic applications and aligns with reported values for Ag–FeBNPs synthesized via green routes³¹.

UV-vis spectra of the phyco-synthesized Ag–FeBNPs capped by Galaxaura rugosa marine algal extract, inset: shows (a) FeCl₃, (b) AgNO₃, (c) G. rugosa extract, and (d)Ag–FeBNPs.

FTIR spectroscopic analysis and XRD analysis of the phyco-synthesized Ag–FeBNPs

The FTIR spectrum of the aqueous extract of G. rugosa (Fig. 3a) exhibits a distinctive peak at 3421 cm⁻¹, attributed to the O-H stretching vibrations within polyphenols and OH groups of sugar rings³². The absorption peaks that appeared at 1598 cm⁻¹ may be due to the vibrations in the C = O bond stretching within the aromatic rings of different phenolic compounds, including polyphenols and flavonoids that are present in extracts of both algal species³³. The spectrum of peaks determined at 1384 cm⁻¹ represents the N = O bond (nitro groups), at 1035 cm⁻¹ represents the C-O stretching of primary alcohols, at 672 cm⁻¹ represents aromatic rings of the C-H bond, and that at 865 cm⁻¹ represents the C-H stretching (vinyl groups)³⁴. The phyco-synthesized Ag–FeBNPs possess various stretching vibrations, as depicted in Fig. 3a. Major peaks appeared at 3421, 1598, 1384, 1035, and 865 cm⁻¹, in correspondence to the stretching of the N-H bond in amines, and O-H in alcohols, C = C bond stretching in alkene, N-O bond stretching in the nitro compound, O-H bond stretching in carboxylic acid, and C = C bond bending in alkenes, respectively. The Ag–FeBNPs showed a minor shift with slight changes, indicating that the main biomolecules present in extracts were capped to the NPs surface as reported by El-Kassas and El Komi³⁵. These shifts confirm that functional groups from bioactive compounds were actively involved in stabilizing the nanoparticles by binding to their surface, forming a protective layer that prevents agglomeration and improves colloidal stability³⁶.

These biomolecules-particularly phenolic compounds, flavonoids, and hydroxyl-rich metabolites-likely acted as dual-function agents: reducing both Ag⁺ and Fe³⁺ ions into their respective metallic states and stabilizing them via capping. The simultaneous reduction of both metal ions nearby may promote the formation of bimetallic nanoparticles either as core-shell structures or alloyed forms, depending on kinetic and thermodynamic factors. The functional groups (-OH, -COOH, -NH₂) facilitate nucleation and prevent agglomeration, enabling the successful integration of Ag and Fe atoms into a single bimetallic nanostructure²⁹.

A comparative looks at other algae-based nanoparticle syntheses further supports these findings. For instance, Sargassum wightii and Ulva lactuca extracts have demonstrated similar FTIR peaks associated with hydroxyl and carbonyl groups that contribute to both metal ion reduction and stabilization^36,37. These similarities affirm the generalizable role of algal biomolecules in green nanoparticle synthesis across species. These results are in good agreement with several previous reports^38,39.

The crystal structure of Ag–FeBNPs is clearly evident in Fig. 3b. The XRD pattern of Ag–FeBNPs from G. rugosa give the diffraction peaks at 2θ values of 28.25°, 29.34°, 31.57°, 40.49°, 46.31°, and 50.31° corresponding to the crystalline planes (210), (220), (104), (114), (231), and (042), emphasizing the face-centered cubic phase of the phyco-synthesized NPs. The diffraction peaks observed in the XRD pattern of Ag–FeBNPs are composed of the standard peaks of Ag (JCPDS no. 04-0783) and Fe (JCPDS no. 33–0664), demonstrating the formation of bimetallic phases of Ag and Fe²⁷.

(a) FTIR of the phyco-synthesized Ag–FeBNPs and G. rugosa marine algal extract; (b) XRD pattern of Ag–FeBNPs capped by G. rugosa marine algal extract.

Morphological analysis of Ag–FeBNPs

The TEM micrograph of Ag–FeBNPs for G. rugosa shows an agglomeration of nano-spheres with particle sizes from 19.95 to 37.11 nm (Fig. 4a) as reported by Kamli, et al.³⁹ who demonstrated the production of spherical Ag–Fe nanoparticles within a range of 100 nm using Beta vulgaris extract. Figure 4b shows the SAED pattern, which exhibits alternating dots and concentric rings, demonstrating that these NPs are polycrystalline⁴⁰.

SEM analyses showed that the particle shapes were spherically shaped, highly distributed, and polydisperse with uniform surfaces (Fig. 4c). These results are in reasonable agreement with previous reports³⁹. The chemical composition of Ag–FeBNPs was securitized upon EDX analysis, as depicted in Fig. 4d. The Fe and Ag signals shown in the EDX spectrum confirmed that these NPs are composed of AgNPs and FeNPs²⁷. Ag–FeBNPs were composed of Fe with 13.51% by weight and 4.31% by atomic percentage, while Ag constituted 14.09% by weight and 2.33% by atomic percentage. These values provide a more precise quantitative assessment of the elemental composition, supporting the successful integration of both metals into the bimetallic structure. The existence of some extra peaks in the spectrum could be attributed to the participation of algal biomolecules in the NPs’ synthesis⁴¹.

(a) TEM, (b) SAED, (c) SEM, and (d) EDX of the phyco-synthesized Ag–FeBNPs capped by G. rugosa marine algal extract.

Zeta potential analysis

Zeta potential, which refers to the degree of stability of the colloidal particles, was used to determine the degree of electrostatic repulsion between adjacent similarly charged particles. The Ag–FeBNPs show negative potential values of 16.1 mV (Fig. 5). This coincides well with previous studies⁴². These negative potential values could be due to the capping impact of the biomolecules present in the algal extracts⁴³. A zeta potential value of −16.1 mV indicates moderate colloidal stability, suggesting that while some electrostatic repulsion is present, aggregation may still occur over time, especially under varying pH or ionic strength conditions. This implies that the nanoparticles may remain sufficiently stable for short-term applications but might require further stabilization for long-term storage or industrial use⁴⁴.

This level of zeta potential suggests that while the nanoparticles are dispersible, their long-term colloidal integrity could be compromised under stress conditions, such as high salinity or variable temperatures, which may limit certain industrial applications without further surface modification⁴⁵. Although this study focused on nanoparticle synthesis and characterization, long-term stability tests and functional performance evaluations (e.g., antimicrobial activity, catalytic efficiency, or adsorption capacity) were not within the scope and are planned for future work.

Zeta potential of Ag–FeBNPs phyco-synthesized from G. rugosa extract.

A comparative analysis with previously reported biosynthesized Ag–FeBNPs using different biological sources, such as fungi and plants, is presented in Table 1. The particle size of Ag–FeBNPs in our study (19.9–37.1 nm) falls within the smaller range compared to fungal-derived nanoparticles, such as those synthesized using Gymnascella dankaliensis (~ 96.8 nm)²⁹. While Gardenia jasminoides-derived Ag–FeBNPs exhibited the smallest size (~ 13 ± 6.3 nm) and core–shell morphology, our nanoparticles displayed a spherical shape and moderate zeta potential of −16.1 mV, indicating acceptable colloidal stability for short-term applications.

Compared to Beta vulgaris-mediated Ag–FeBNPs (~ 15 nm), which showed antifungal and apoptosis-inducing activities³⁹, the G. rugosa–derived nanoparticles remain primarily characterized, with functional applications such as antimicrobial or catalytic assessments planned for future work. Additionally, while Gardenia jasminoides-synthesized Ag–FeBNPs demonstrated magnetic behavior and broad-spectrum antimicrobial potential⁴⁶, our synthesis emphasizes eco-friendliness and ease of preparation. These comparisons highlight the uniqueness of using red marine algae as a sustainable and efficient route for bimetallic nanoparticle biosynthesis.