UV photoluminescence and antimicrobial properties of silver ion modified calcined and non calcined eggshell based substrates

Abstract

In this study, raw and calcined eggshell-based biomaterials were modified with Ag⁺ ions, and their structural, surface, optical, and antimicrobial properties were systematically investigated. A sustainable approach was used to valorize eggshell waste, with AgNO₃ providing Ag-related species immobilized onto the material surface. X-ray diffraction confirmed the transformation of CaCO₃ to CaO upon calcination, while the absence of metallic silver peaks indicated incorporation of Ag⁺ within the nanocomposite. Zeta potential measurements showed increased positive surface charge after Ag⁺ modification, particularly in calcined samples, suggesting enhanced surface reactivity and colloidal stability. EDX analysis revealed localized Ag accumulation on non-calcined eggshells, whereas calcined composites exhibited more homogeneous Ag distribution. Photoluminescence studies showed green emission for ES@Ag⁺, while PL intensity was suppressed in C-ES@Ag⁺ due to Ag⁺-induced surface defects and charge transfer. Ag⁺ emission confirmed by UV-Vis. In addition, minimum inhibitory concentration (MIC) analysis revealed that ES@Ag⁺ exhibited superior antimicrobial efficiency at lower concentrations compared to C-ES@Ag⁺, demonstrating consistent performance across both liquid and solid media. Antimicrobial activity was tested against Candida albicans ATCC 10239 and Escherichia coli ATCC 8739 via the agar well diffusion method. ES@Ag⁺ exhibited inhibition zones of 17.00 ± 0.05 mm and 16.00 ± 0.07 mm, while C-ES@Ag⁺ showed 15.00 ± 0.02 mm and 9.00 ± 0.05 mm for C. albicans and E. coli, respectively. These results demonstrate the potential of Ag⁺-modified eggshell nanocomposites as sustainable materials for biomedical and environmental applications. While antimicrobial efficacy is promising, further cytotoxicity and biocompatibility studies are needed to assess safety and performance.

Graphical abstract

Introduction

In recent years, increasing environmental concerns have intensified the importance of sustainable approaches aimed at converting biological wastes into value-added functional materials. In this context, eggshells, which attract attention due to their high calcium content and natural origin, have emerged as low-cost and environmentally friendly raw materials for the development of biomaterial-based systems [1, 2]. Calcium carbonate (CaCO₃), the main component of eggshells, can be transformed into different crystalline phases through appropriate thermal and chemical treatments, and these phase transformations play a decisive role in surface chemistry and functional properties [3–5]. It is well established that the CaCO₃ structure is converted into CaO when eggshells are subjected to high-temperature calcination [5]. This transformation alters surface alkalinity and the nature of active sites, thereby directly affecting ion adsorption, surface charge characteristics, and interactions with microorganisms [6, 7]. Therefore, a comparative investigation of calcined and non-calcined eggshell surfaces is of great importance for the rational design of functional biomaterials.

Silver ions (Ag⁺) are among the widely used active species in biomedical and environmental applications due to their broad-spectrum antibacterial and antifungal properties [8, 9]. The interaction of Ag⁺ ions with cell wall proteins, disruption of membrane permeability, induction of reactive oxygen species (ROS) generation, and suppression of cellular metabolism constitute the primary antimicrobial mechanisms of these ions [10–12]. However, the uncontrolled release and potential toxic effects of metallic silver nanoparticles necessitate the development of alternative systems in which silver-containing species (e.g. Ag⁺ ions) are immobilized on biocompatible surfaces.

The novelty of this work lies in its simplicity and sustainability. In contrast to many studies that employ multi-step synthesis routes, stabilizing agents, or chemical reducing processes, this study demonstrates a straightforward approach based on the direct interaction between eggshell-derived matrices and silver nitrate. The results suggest that the intrinsic surface chemistry of raw and calcined eggshells can facilitate the immobilization and stabilization of silver-related species without the need for additional reagents. This enables the conversion of a common biowaste into a functional material exhibiting both antimicrobial activity and photoluminescence behavior. The combination of low-cost processing, waste valorization, and multifunctional properties highlights the potential of this system as a promising platform for sustainable biomaterial design.

Beyond their antimicrobial activity, Ag⁺-modified biomaterials are also of considerable interest due to their optical behavior. In particular, surface defects and ionic centers formed by the incorporation of Ag⁺ ions into amorphous or low-crystallinity structures can give rise to broad photoluminescence (PL) emission bands in the visible region [13–15]. PL analyses provide valuable insights into ion–matrix interactions, charge transfer processes, and surface defect states, thereby contributing to a deeper understanding of the optical performance of functional materials [16, 17]. Investigating the distribution of Ag⁺ ions on calcined and non-calcined eggshell surfaces and correlating their effects on both PL behavior and antimicrobial performance is expected to provide a significant contribution to the existing literature.

PL behavior of eggshell-derived materials is highly dependent on the crystalline structure and the preservation of the biogenic organic-inorganic interface. Pure, synthetic CaCO₃ is a wide-bandgap insulator that typically does not exhibit intrinsic visible luminescence [18]. However, biomass-derived eggshells contain a complex organic matrix, including residual proteins and membranes, which may provide functional anchoring sites for the stable immobilization of silver-related species. The interaction between these silver species and the protein-rich surface is likely to introduce localized electronic states and active radiative recombination centers, contributing to distinct PL emissions in the CaCO₃ system [19–21]. In contrast, high-temperature calcination leading to CaO formation results in the thermal degradation of these organic constituents. This structural transformation, together with the more homogeneous distribution of silver species, may limit the formation of localized luminescent centers or defect states typically responsible for radiative recombination. Consequently, the PL intensity observed in calcined samples is significantly reduced compared to the non-calcined system.

In this study, raw and calcined eggshell substrates were modified with Ag⁺ ions to synthesize ES@Ag⁺ and C-ES@Ag⁺ nanocomposites, and the effects of the calcination process on their structural, surface, optical, and antimicrobial properties were comparatively examined. The obtained nanocomposites were characterized by XRD, EDX, zeta potential, and photoluminescence analyses, and their antimicrobial activities were evaluated against Candida albicans ATCC 10239 and Escherichia coli ATCC 8739. This work aims to demonstrate that eggshell waste can be transformed into sustainable, biocompatible, and multifunctional biomaterials through functionalization with Ag⁺ ions.

Materials and methods

Materials

In this study, Mueller–Hinton Broth (MHB), Mueller–Hinton Agar (MHA), Tryptic Soy Agar (TSA), and Tryptic Soy Broth (TSB) were purchased from Biolife Italiana (Italy). Silver nitrate (AgNO₃, extra pure, Sigma 209139) and chlorhexidine acetate (CHX, Fluka Analytical, PHR1222) were obtained from Fluka Analytical. R7017 Sigma-Aldrich Resazurin sodium salt was used in the Minimum Inhibitory Concentration test. All solutions and culture media were prepared using ultrapure water (R ≥ 18.3 MΩ·cm) produced by a Zener 900 water purification system (Human Corporation, Korea).

Methods

Preparation of eggshell powder

In this study, commercial eggshells were processed into powder through a series of pretreatment steps. The collected eggshells were first washed with tap water to remove surface contaminants, followed by three rinses with distilled water, and then allowed to dry at room temperature. After drying, ultrapure water was added to the shells, and they were boiled by heating at a set temperature of 125 °C for 2 h under ambient pressure. Following the boiling process, the shells were filtered and again dried at room temperature. The dried eggshells were ground into a fine powder using an agate mortar and stored in a desiccator for subsequent use.

Calcination of eggshells

For the calcination process, eggshell powders were placed in ceramic crucibles and transferred to a muffle furnace. The samples were subjected to thermal treatment in air at 900 °C for 3 h with a heating rate of 10 °C/min to ensure the thermal decomposition of CaCO₃ into CaO. After completion of the calcination process, the furnace was allowed to cool naturally to room temperature. The calcined eggshells exhibited an approximate weight loss of 75%, yielding calcined eggshell powder predominantly composed of CaO. The obtained calcined powders were stored in a desiccator until further use.

Eggshell mediated nanocomposite synthesis

The silver modification process was carried out by maintaining a solid-to-liquid ratio of 1:5 (g/mL), 25 mL of 0.01 M AgNO₃ solution was added to 5 g of both calcined and non-calcined eggshell powders in two separate beakers. The resulting suspensions were continuously stirred at 1400 rpm for 7 h. Subsequently, the solutions were left to stand at room temperature (25 °C) overnight. After incubation, the suspensions were centrifuged at 5000 rpm for 4 min. The supernatants were discarded, and the precipitates were washed three times with ultrapure water. The samples were then transferred to ceramic crucibles and dried in an oven at 60 °C for 20 h (Fig. 1). The named eggshell powders, calcined eggshell powders and the synthesized nanocomposites were designated as listed in Table 1.

Fig. 1.

Synthesis scheme of ES@Ag⁺ and C-ES@Ag⁺ nanocomposites

Table 1.

Naming and composition of nanocomposites (ES: Eggshell)

Nanocomposites	Market ES	Calcined Market ES	0.01 M AgNO3
ES@Ag+	+	−	+
C-ES@Ag+	−	+	+

Characterizations

The surface morphology of the eggshells and nanocomposite samples was examined using a ZEISS SUPRA 40VP GEMINI scanning electron microscope (SEM). Prior to analysis, the samples were coated with a thin layer of platinum using a QUORUM Q ISORES sputter coater to enhance electrical conductivity. X-ray diffraction (XRD) measurements were performed to identify the crystalline structures using Cu Kα radiation with a wavelength of λ = 1.5406 Å, and the diffraction patterns were recorded in the 2θ range of 10°–90°. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectra were recorded in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ to determine the functional groups present in the nanocomposites. Zeta potential measurements of the samples were carried out using a Malvern Nano ZS instrument.

Photoluminescence evaluation of samples under UV light

For photoluminescence evaluation, 2.0 g of each sample, including calcined and non-calcined eggshells as well as ES@Ag⁺ and C-ES@Ag⁺ nanocomposites, were dispersed in 10 mL of ultrapure water. The resulting colloidal suspensions were magnetically stirred until a homogeneous dispersion was obtained. The prepared suspensions were examined under UV-A light to qualitatively evaluate their photoluminescence properties. Photoluminescence behavior was recorded based on the observed color changes and emission intensity of the samples during UV light exposure [17].

Photoluminescence (PL) analysis

Room-temperature PL spectroscopy measurements were carried out to evaluate the optical emission behavior and defect states of Market ES and Calcined Market ES eggshell powders and their silver-modified nanocomposites, ES@Ag⁺ and C-ES@Ag⁺. PL spectra were recorded using an ANDOR SR500i-BL luminescence spectrometer equipped with a triple grating monochromator and an intensified charge-coupled device (ICCD) detector for the visible region. A frequency tripled Nd: YLF Q-switched pulsed laser operating at a wavelength of 349 nm was used as the excitation source. During PL measurements, the samples were analyzed directly in solid powder form without the use of any solvent. Emission spectra were recorded in the wavelength range of 400–700 nm covering the visible region. For comparison of emission characteristics among different samples, the obtained spectral data were normalized.

UV–vis spectroscopic analysis

The optical absorption properties of the synthesized nanocomposites were analyzed using a Thermo Scientific Genesys 150 UV–Vis spectrophotometer. Market ES was used as the blank for ES@Ag⁺, while Calcined Market ES was used as the blank for C-ES@Ag⁺ to isolate the absorption features of the immobilized silver species. The spectra were recorded in the wavelength range of 280–500 nm with a scanning resolution of 2 nm. This analysis aimed to elucidate the oxidation state of the silver species and to identify the electronic transitions associated with Ag⁺ ions. The absence of absorption bands in the 400–500 nm region was used to confirm the lack of surface plasmon resonance (SPR), indicating that metallic silver nanoparticle (AgNP) formation did not occur.

Determination of minimum inhibitory concentration (MIC)

The Minimum Inhibitory Concentration (MIC) of the synthesized samples was determined using the resazurin-based microdilution method against Escherichia coli ATCC 8739, and Candida albicans ATCC 10239. The microorganisms were cultured overnight at 37 °C in Mueller-Hinton Broth (MHB) and subsequently adjusted to a final concentration of 5 × 10⁵ CFU/mL. The assays were performed in sterile 96-well microplates. Initially, 100 µL of MHB medium was added to each well. Stock solutions (4000 ppm) of Market ES, Calcined Market ES, ES@Ag⁺, and C-ES@Ag⁺ were prepared in sterile distilled water. A 100 µL aliquot of the respective stock solution was added to the first row and serially diluted twofold from top to bottom (resulting in a concentration range from 2000 to 62.5 ppm). Following the dilution, each well was inoculated with 10 µL of the microbial suspension. To visualize metabolic activity, 10 µL of resazurin solution (6.75 mg/mL) was added to each well as a redox indicator. The plates were incubated at 37 °C for 24 h. Two sets of controls were maintained: a positive control consisting of MHB, microbial culture, and resazurin (to confirm microbial viability), and a negative control containing only MHB and resazurin (to ensure medium sterility). To ensure reproducibility, each concentration was tested in triplicate (n = 3) using three adjacent wells. The MIC was defined as the lowest concentration of the sample that prevented the color change of resazurin from purple to pink [22].

Antibacterial test

The antibacterial activities of Calcined Market ES, C-ES@Ag⁺, Market ES, and ES@Ag⁺ samples were evaluated against Escherichia coli ATCC 8739 using the agar well diffusion method. E. coli ATCC 8739 was inoculated into Tryptic Soy Broth (TSB) from a stock culture and incubated at 37 °C for 24 h. After incubation, the bacterial suspension was diluted to a concentration of 1.5 × 10⁶ CFU/mL and spread onto Tryptic Soy Agar (TSA) plates using a sterile swab. Aliquots of 40 µL from 1% (w/v) sample solutions prepared in sterile ultrapure water were placed into 6 mm well diameter formed using a sterile borer. Chlorhexidine acetate (CHX) was used as the positive control. The plates were incubated at 37 °C for 24 h. After incubation, the antibacterial activities of the samples were evaluated by measuring the diameters of the inhibition zones around the wells in millimeters (mm) [23, 24]. The agar well diffusion tests were conducted in three independent replicates, and the inhibition zones were calculated as mean ± standard deviation.

Antifungal test

The antifungal activities of Calcined Market ES, C-ES@Ag⁺, Market ES, and ES@Ag⁺ samples against Candida albicans ATCC 10239 were evaluated using the agar well diffusion method. Candida albicans from a stock culture was inoculated into Mueller Hinton Broth (MHB) and incubated at 37 °C for 24 h. The resulting suspension was adjusted to a concentration of 1.5 × 10⁶ CFU/mL and spread onto Mueller Hinton agar plates (prepared according to CLSI M44-A2 standards: Mueller–Hinton agar supplemented with 2% glucose and 0.5 µg/mL methylene blue) using a sterile swab. Aliquots of 40 µL from 1% (w/v) sample solutions prepared in sterile ultrapure water were placed into 6 mm well diameter formed using a sterile borer. CHX was used as the positive control. The agar plates were incubated at 37 °C for 48 h. After incubation, the antifungal activities of the samples were evaluated by measuring the diameters of the inhibition zones around the wells in mm [23, 24]. The agar well diffusion tests were conducted in three independent replicates, and the inhibition zones were calculated as mean ± standard deviation.

Results

The SEM images of Market ES, Calcined Market ES, ES@Ag⁺, and C-ES@Ag⁺ samples are presented in Fig. 2. The SEM image of the eggshells obtained from the market reveals the presence of micro-nano pores and calcite structures on the surface (Fig. 2a). After calcination of the eggshells at 900 °C, the SEM images of the Calcined Market ES sample show the deterioration of the micro–nano porous structure inherent to the raw eggshell and the formation of CaO structures (Fig. 2b). This observation is consistent with the literature, where numerous studies have reported the transformation of the CaCO₃ structure into the CaO phase upon high-temperature calcination of eggshells [5, 25, 26]. The SEM image of the ES@Ag⁺ indicates that the pores and surface of the eggshell are covered with Ag-related structures, which are locally aggregated (Fig. 2c). Similarly, in the C-ES@Ag⁺, Ag⁺ species are observed to accumulate on the surface of CaO structures, although to a lesser extent compared to the ES@Ag⁺ nanocomposite (Fig. 2d).

Fig. 2.

SEM images of eggshell samples: a Market ES, b Calcined Market ES, c ES@Ag⁺, and d C-ES@Ag⁺

The EDX spectra of the nanocomposite samples are presented in Fig. 3 and their corresponding elemental weight percentages are detailed in Table 2. For the ES@Ag⁺ nanocomposite, EDX analyses were performed on two different regions. A comparison of the data obtained from the eggshell surface (spectrum 7377) and from the Ag rich clusters (spectrum 7376) indicates that the elemental Ag content (39.85%) is significantly higher in the Ag clusters accumulated on the surface (Fig. 3a). In contrast, for the C-ES@Ag⁺ nanocomposite, comparison of the spectra acquired from two different regions of the calcined eggshell surface (spectra 6185 and 6186) reveals a very low elemental Ag content (0.23%) (Fig. 3b). These findings demonstrate that Ag⁺ ions can be immobilized on both calcined and non calcined eggshell surfaces; however, due to differences in surface morphology and the chemical and elemental composition altered by the calcination process, the amount of silver bound to the surface and its distribution pattern vary significantly. The presence of a platinum (Pt) signal at around 2.1 keV in the EDS results is a direct consequence of the conductive Pt coating process performed prior to SEM imaging.

Fig. 3.

SEM–EDX spectra of the nanocomposites: a ES@Ag⁺ and b C-ES@Ag⁺

Table 2.

EDX spectrum results of nanocomposites

Sample	Element	Weight (wt%)	Atomic (at%)	Error (wt%)
ES@Ag⁺ Spectrum: 7376	Silver (Ag)	39.85	11.37	1.21
	Oxygen (O)	30.41	58.48	6.91
	Calcium (Ca)	25.64	19.68	0.90
	Carbon (C)	4.09	10.47	1.49
ES@Ag⁺ Spectrum: 7377	Calcium (Ca)	57.86	35.34	1.40
	Oxygen (O)	38.27	58.55	9.02
	Silver (Ag)	0.97	0.22	0.15
	Carbon (C)	2.89	5.89	1.47
C-ES@Ag⁺ Spectrum: 6185	Calcium (Ca)	57.46	34.76	1.62
	Oxygen (O)	41.01	62.14	8.70
	Carbon (C)	1.53	3.10	0.86
C-ES@Ag⁺ Spectrum: 6186	Calcium (Ca)	49.91	28.26	1.32
	Oxygen (O)	47.75	67.72	8.40
	Carbon (C)	2.10	3.97	0.91
	Silver (Ag)	0.23	0.05	0.07

The presence of Ag⁺ ions in their most stable oxidation state in aqueous environments and their ability to remain stable in solution facilitate their adsorption onto various inorganic surfaces [27–29]. In this context, the carbonate (CO₃²⁻) groups present in the CaCO₃-dominant structure of non-calcined eggshells provide favorable binding sites for Ag⁺ ions, leading to their localized accumulation and enrichment at specific surface regions. In addition, the organic eggshell membrane, which is rich in functional groups such as amine, carboxyl, and thiol moieties, offers preferential binding sites for Ag⁺ ions, contributing to the localized retention of silver and its concentration in the form of crystalline-like deposits.

This surface-binding behavior is consistent with previous studies reporting strong interactions between Ag⁺ ions and functional groups on substrate surfaces. In particular, the tendency of Ag⁺ ions to form complexes with negatively charged surfaces and ligands has been shown to significantly influence surface adsorption processes [29–32]. The transformation of the CaCO₃ structure into the CaO phase through calcination results in the removal of organic membrane components and induces significant changes in surface chemistry. Hydroxyl (–OH) groups present on CaO surfaces promote a more homogeneous and dispersed immobilization of Ag⁺ ions [33–35]. The affinity of Ag⁺ ions for oxygen-containing ligands favors a uniform surface distribution rather than localized accumulation, which is consistent with the low but homogeneous Ag content observed in the EDX analyses of the C-ES@Ag⁺ nanocomposite [36, 37]. In the calcined system, the absence of sulfur and thiol containing ligands renders Ag⁺ binding through surface oxygenated groups the dominant immobilization mechanism. Consequently, the changes in surface morphology and chemical composition induced by calcination play a decisive role in governing the adhesion and distribution behavior of Ag⁺ ions on the eggshell surface.

XRD analysis was performed to determine the crystalline structures of the synthesized nanocomposites and the calcined and non-calcined eggshell powders. According to the library matching of the XRD patterns shown in Fig. 4a, the characteristic diffraction peaks observed at 2θ = 23.14°, 29.47°, 36.04°, 39.46°, 47.55°, 48.54°, and 48.68° for the non-calcined Market ES sample were assigned to the calcite CaCO₃ phase (CaCO₃, card no. 96-901-6023). The XRD diffraction pattern of the ES@Ag⁺ sample exhibited a profile similar to that of Market ES, indicating the dominance of the CaCO₃ phase. The absence of characteristic diffraction peaks corresponding to metallic silver Ag⁰ confirms that silver did not form in a crystalline metallic state. This observation suggests that silver was adsorbed onto the eggshell surface primarily as Ag-related species with a dominant ionic character (Ag⁺) and likely bound as Ag–O–Ca species on the surface.

Fig. 4.

a XRD diffraction patterns and b zeta potential data of the samples

The XRD diffraction pattern of Calcined Market ES revealed a clear deviation from the calcite structure, with diffraction peaks observed at 2θ = 18°, 28.7°, 34.1°, 50.8°, 54.4°, and 62.5°, which were assigned to the CaO phase (CaO, card no. 96-900-9099). After the calcination process, a small portion of the CaCO₃ structure is converted into calcium hydroxide (Ca(OH)₂). In the XRD patterns, minor peaks corresponding to Ca(OH)₂ can be observed at approximately 2θ = 28° and 54°. The formation of Ca(OH)₂ during calcination is a common phenomenon and has been widely reported by several researchers during the synthesis of CaO [38]. This transformation indicates that the calcite structure of the eggshell was converted into CaO upon high-temperature calcination, as described by Eq. (1), leading to a significant change in the crystalline structure [39, 40].

1

In the XRD diffraction pattern of the C-ES@Ag⁺ nanocomposite, the characteristic diffraction peaks of the CaO phase were preserved, while no distinct peaks corresponding to metallic silver were observed. This indicates that silver was immobilized on the surface of the calcined eggshell as Ag-related species (including ionic and amorphous forms), forming ionic or amorphous Ag species on the surface. The absence of XRD detectable Ag related peaks is an expected result, considering the low silver content and the lack of long-range crystalline order in such surface-bound Ag⁺ species [41–43].

To evaluate the surface charge characteristics and colloidal stability of the samples, zeta potential measurements were performed (Fig. 4b). The measured pH values of the colloidal suspensions during zeta potential analysis (Table 3) showed a highly alkaline nature (pH ~ 12.3–12.9). This behavior can be attributed to the hydration of CaO into Ca(OH)₂ upon contact with the aqueous medium, a process known to induce structural changes and expose fresh active surfaces [44–46]. Under such alkaline conditions, surface hydroxyl groups may undergo partial deprotonation, leading to the formation of negatively charged surface sites. These sites provide a favorable electrochemical environment for the electrostatic interaction and stabilization of positively charged silver species, as commonly reported for hydroxylated oxide surfaces [46]. The positive shift in zeta potential observed in Table 3 (Fig. 4b) is consistent with the association of positively charged silver species with the hydrated eggshell matrix. The non-calcined Market ES exhibited a negative surface charge with a zeta potential value of − 18.53 ± 0.51 mV, which can be attributed to the dominant influence of carbonate groups present on the CaCO₃ surface [47, 48]. For the ES@Ag⁺ sample, the zeta potential increased to − 14.80 ± 0.56 mV, indicating a less negative surface compared to Market ES. This shift is attributed to the adsorption of silver onto the eggshell surface as Ag-related species with a dominant ionic character rather than metallic nanoparticles. Electrostatic interactions between Ag⁺ ions and carbonate groups on the CaCO₃ surface, along with the formation of Ag–O–Ca surface complexes, increase the surface charge density and shift the zeta potential toward more positive values [49, 50].

Table 3.

pH and zeta potential (mV) values of the samples measured during zeta potential analysis

Sample	pH value	Zeta potential (mV)
Market ES	12.28	−18.53 ± 0.51
Calcined Market ES	12.90	8.45 ± 0.60
ES@Ag+	12.10	−14.80 ± 0.56
C-ES@Ag+	12.77	14.20 ± 0.61

For the calcined Market ES sample, a positive zeta potential value of 8.45 ± 0.60 mV was obtained, which is attributed to the alkaline surface structure resulting from the partial conversion of CaO into Ca(OH)₂ in aqueous environments [51–53]. In the C-ES@Ag⁺ nanocomposite, and calcined eggshell powder, the zeta potential further increased to 14.20 ± 0.61 mV, indicating a significantly higher positive surface charge compared to the non-calcined system. This behavior can be explained by the enhanced adsorption of Ag⁺ ions on the CaO/Ca(OH)₂ surface under high-pH conditions. The immobilization of Ag⁺ ions increases the positive surface charge density, resulting in a pronounced positive shift in the zeta potential [49, 50]. When the absence of metallic silver peaks in the XRD analysis and the positive shift in zeta potential after Ag modification are considered together, it can be concluded that silver was incorporated into the nanocomposite structure in its ionic Ag⁺ form, forming surface bound Ag species with an amorphous character.

The surface functional groups and changes in the chemical composition of the prepared samples were investigated by FTIR spectroscopy. The obtained spectra are presented in Fig. 5, and the characteristic band positions are summarized in Table 4. In both Market ES and ES@Ag⁺ samples, the characteristic vibrational modes of CaCO₃ corresponding to the most stable polymorph, calcite, are predominantly observed. In particular, the intense and broad band centered around ~ 1420 cm⁻¹ is attributed to the asymmetric stretching vibration of carbonate groups, while the sharp peaks at approximately 874 cm⁻¹ and 712 cm⁻¹ further confirm the presence of carbonate species [12, 54, 55]. These findings indicate that the fundamental inorganic structure of the eggshell is preserved during the synthesis process. The effect of calcination was further evaluated by comparing calcined and non-calcined samples. In the non Calcined Market ES and ES@Ag⁺ samples, aliphatic C–H stretching vibrations in the range of 2980–2875 cm⁻¹ and the amide I band centered around ~ 1645 cm⁻¹, originating from organic components of the eggshell membrane such as proteins and collagen, were clearly detected [56, 57]. In contrast, these bands significantly decreased in intensity or completely disappeared in the calcined samples (Calcined Market ES and C-ES@Ag⁺) as a result of thermal treatment, indicating the effective removal of the organic matrix [5, 58]. Additionally, weak bands observed in all spectra within the range of 3600–3640 cm⁻¹ were assigned to the free stretching vibrations of hydroxyl (–OH) groups from physically adsorbed water molecules and the residual organic components (proteins/collagen) present in the uncalcined biogenic matrix [24, 56]. While this peak is often associated with Ca(OH)₂ in calcined samples, its presence in the Market ES indicates the hydration of the organic-inorganic interface. Upon the incorporation of silver species into the structure, slight shifts in the main characteristic bands were observed, which may indicate interactions between silver-containing species and the functional groups present on the eggshell surface [59, 60]. These subtle infrared features, together with the observed shifts in zeta potential, may indicate the presence of surface-level interactions contributing to the stabilization of silver-containing species.

Fig. 5.

FTIR spectra of non calcined and calcined eggshell based samples before and after Ag⁺ immobilization

Table 4.

FTIR peak positions and corresponding functional group assignments before and after synthesis

Functional Group	Calcined Market	C-ES@Ag⁺	Market ES	ES@Ag⁺
O–H	3269, 3642	3638	3617	3641
C–H	2976, 1093, 1052	2925	2980, 2875	2981, 2878
C=O	1685	1650	1645	1642
CaCO₃	2530, 1422, 874, 712	2516, 1418, 874, 712	2516, 1418, 875, 712	2515, 1420, 874, 712

The PL spectra of the four materials were recorded in the wavelength range of 400–700 nm under a constant excitation of 349 nm, and the results are presented in Fig. 6a. Market ES and Calcined Market ES samples exhibited very low and nearly constant PL intensities throughout the measured range, indicating that CaCO₃ and CaO matrices do not possess distinct optical emission centers. In contrast, the ES@Ag⁺ nanocomposite displayed a broad and intense emission band with a maximum in the range of approximately 480–520 nm. This enhanced PL response is likely associated with Ag⁺-related surface states, defect centers, and charge transfer interactions arising from the incorporation of silver ions into the CaCO₃ matrix and their interaction with functional groups present in the eggshell structure, such as amide and thiol groups [61–63]. Numerous studies have reported that protein metal cluster interactions can induce photoluminescent behavior, supporting this interpretation [64–67]. In particular, surface complexes formed between Ag⁺ ions and carbonate groups, as well as short range ordered Ag containing clusters may increase the probability of radiative recombination, resulting in a strong PL response [68]. However, further spectroscopic analyses, such as excitation-dependent PL measurements and lifetime studies, would be required to conclusively determine the origin of the emission. In contrast, the PL intensity of the C-ES@Ag⁺ nanocomposite prepared using calcined eggshell was significantly suppressed. This behavior may be attributed to the structural transformation CaCO₃ structure, the removal of the organic membrane after calcination, and the more homogeneous dispersion of Ag⁺ ions on the surface without localized clustering, leading to the dominance of non-radiative relaxation pathways. Furthermore, the reduced carbonate content after calcination weakens Ag⁺–CO₃²⁻ interactions, potentially decreasing the density of PL-active sites [69, 70]. Photographs of the sample under daylight and UV illumination are shown in Fig. 6b, where a green emission is observed the ES@Ag⁺ nanocomposite under UV light, in good agreement with the PL results.

Fig. 6.

Effect of Ag⁺ incorporation into raw and calcined eggshell matrices on optical properties: a Photoluminescence (PL) spectra of four samples recorded in solid powder form under excitation at 349 nm and b photographs under daylight and UV illumination (365 nm), and c UV–Vis absorption spectra showing Ag⁺-related charge transfer bands and confirming the absence of surface plasmon resonance (SPR) bands associated with metallic silver nanoparticles

The electronic transition mechanism and the nature of the luminescent centers were further elucidated by correlating the absorption properties with the PL response (Fig. 6c). The UV–Vis spectra exhibit a distinct absorption band at ~ 290 nm, which can be attributed to the electronic excitation of surface-immobilized Ag⁺ ions through charge-transfer interactions with the substrate [71]. Importantly, the absence of a localized surface plasmon resonance (LSPR) band in the 400–500 nm region indicates that silver is predominantly present in its ionic form (Ag⁺) rather than as metallic nanostructures [72, 73]. The excitation of these ionic states under UV illumination is likely responsible for the green emission (480–520 nm) observed in the PL spectra. This correlation suggests that Ag⁺ ions, stabilized by the biogenic functional groups of the eggshell matrix, act as the primary radiative recombination centers, as metal ion–ligand interactions are known to induce charge-transfer transitions in the UV region, which can subsequently lead to photoluminescence emission [74] The suppressed PL response in the C-ES@Ag⁺ system further supports this interpretation, as the removal of organic components and partial loss of carbonate groups during calcination reduce the density of photoactive sites.

The antimicrobial activities of Market ES, Calcined Market ES, and silver-modified ES@Ag⁺ and C-ES@Ag⁺ were colorimetric evaluated using a resazurin-based microdilution assay. This colorimetric MIC method relies on a redox indicator that reflects microbial metabolic activity. Viable cells reduce blue/purple resazurin to pink resorufin through intracellular electron transfer processes associated with cellular respiration. Therefore, the absence of a color change indicates effective inhibition of microbial growth, whereas a shift to pink confirms metabolic activity and cell viability [75]. The visual results demonstrating microbial metabolic transitions are presented in Fig. 7.

Fig. 7.

Minimum inhibitory concentration (MIC) assay results of Market ES, Calcined Market ES, ES@Ag⁺, and C-ES@Ag⁺ against C. albicans ATCC 10239 and E. coli ATCC 8739

As observed in the microplate assays, Market ES exhibited no intrinsic inhibitory effect against either E. coli and C. albicans, as evidenced by the rapid color transition from purple to pink across all tested concentrations (2000 to 62.5 ppm). This finding confirms that the pure CaCO₃ matrix does not possess inherent biocidal properties at the tested concentrations [22]. In contrast, the ES@Ag⁺ nanocomposite showed a significant enhancement in antimicrobial activity due to the presence of silver-derived structures, achieving an MIC value of 125 ppm against E. coli and 250 ppm against C. albicans. This pronounced antimicrobial effect can be attributed to the controlled and sustained release of Ag⁺ ions, facilitated by the high surface area of the porous eggshell structure. The released Ag⁺ ions interact electrostatically with the microbial cell wall and membrane, disrupting membrane permeability and causing leakage of intracellular contents. Additionally, Ag⁺ ions induce the generation of ROS, leading to oxidative stress, protein denaturation, DNA damage, and inhibition of enzymatic systems, ultimately resulting in cell death [76, 77].

An interesting observation was recorded for the Calcined Market ES sample. Despite the absence of silver, Calcined Market ES sample exhibited notable growth inhibition, particularly at higher concentrations. A concentration of 62.5 ppm was sufficient for inhibition of both microorganisms. This phenomenon can be attributed to the structural transformation of CaCO₃ into CaO during calcination at 900 °C [5]. Upon contact with the aqueous culture medium, CaO undergoes hydration to form Ca(OH)₂, resulting in a significant increase in local pH. This highly alkaline environment, combined with the generation of ROS on the CaO surface, creates lethal conditions for most microorganisms [78]. Previous studies by Thanakkasaranee and Seo (2020) have demonstrated that the elevated alkalinity and ROS production associated with CaO disrupt bacterial cell wall integrity, leading to cell death. Furthermore, Ca²⁺ ions released from the CaO structure can interact with cardiolipin molecules in bacterial membranes, destabilizing membrane structure and promoting leakage of intracellular components [79]. These mechanisms collectively explain the inhibitory effect observed for calcined samples even in the absence of silver.

In contrast, the C-ES@Ag⁺ nanocomposite exhibited higher MIC values of 250 ppm for C. albicans and 1000 ppm for E. coli, indicating a reduction in antimicrobial activity compared to the silver-free Calcined Market ES. This observation suggests that the expected synergistic effect is not achieved; instead, a partial antagonistic interaction may occur within the system. This behavior can be attributed to the strong interactions between Ag⁺ ions and the CaO surface formed after calcination. Specifically, the high affinity of Ag⁺ ions for the CaO surface may lead to partial passivation of active sites, thereby limiting the characteristic antimicrobial mechanisms of CaO, such as high alkalinity and ROS generation. Moreover, the strong adsorption of Ag⁺ ions may reduce their diffusion into the surrounding medium and limit their bioavailability, preventing the attainment of effective concentrations during short-term MIC assays. In addition, electrostatic interactions between the positively charged CaO surface and Ag⁺ ions may create a kinetic barrier that hinders the transfer of silver species toward negatively charged microbial cell membranes. Furthermore, structural changes in porosity and surface morphology after calcination may impair the homogeneous distribution of silver, reducing the effective contact area. Collectively, these factors result in restricted ion mobility and diminished antimicrobial performance in the C-ES@Ag⁺ system [80].

The antimicrobial activities of raw and calcined eggshells and Ag⁺ loaded ES@Ag⁺ and C-ES@Ag⁺ nanocomposite samples were evaluated using the agar well diffusion method. Representative images of the inhibition zones are presented in Fig. 8, and the corresponding inhibition zone diameters are summarized in Table 5. Antimicrobial assays were performed against Candida albicans ATCC 10239 and Escherichia coli ATCC 8739. CHX was used as a reference antimicrobial agent and served as the positive control.

Fig. 8.

Antimicrobial activity of eggshell-based samples before and after Ag⁺ immobilization evaluated by agar well diffusion assay against C. albicans ATCC 10239 and E. coli ATCC 8739

Table 5.

Mean inhibition zone diameters (mm) ± standard deviation of eggshell-based samples against C. albicans ATCC 10239 and E. coli ATCC 8739

Samples	Candida albicans ATCC 10,239	Escherichia coli ATCC 8739
CHX	23.00 ± 0.05	17.00 ± 0.06
Market ES	–	–
Calcined Market ES	9.00 ± 0.03	–
ES@Ag⁺	17.00 ± 0.05	16.00 ± 0.07
C-ES@Ag⁺	15.00 ± 0.02	9.00 ± 0.05

According to the results, the Market ES sample did not produce any inhibition zones against either microorganism, indicating that raw eggshell alone does not exhibit significant antimicrobial activity. The Calcined Market ES sample produced an inhibition zone with a diameter of 9 ± 0.03 mm against C. albicans, while no inhibitory effect was observed against E. coli. This finding suggests that the CaO/Ca(OH)₂ structure formed after calcination may exert a limited antifungal effect but is insufficient to provide antibacterial activity against Gram negative bacteria. In contrast, a pronounced enhancement in antimicrobial activity was observed for the Ag⁺ modified samples. The ES@Ag⁺ nanocomposite generated inhibition zones of 17 ± 0.05 mm and 16 ± 0.07 mm in diameter against C. albicans and E. coli, respectively. Similarly, the C-ES@Ag⁺ nanocomposite produced inhibition zones of 15 ± 0.02 mm against C. albicans and 9 ± 0.05 mm against E. coli. The positive control CHX formed inhibition zones of 23 ± 0.05 mm and 17 ± 0.06 mm against C. albicans and E. coli, respectively, enabling a direct comparison between the synthesized nanocomposites and a widely used, potent antimicrobial agent [81].

Notably, the antibacterial activity of ES@Ag⁺ against E. coli was found to be comparable to that of CHX, while its antifungal activity against C. albicans was lower than CHX but still considerable. The high antimicrobial performance exhibited by the ES@Ag⁺ nanocomposite can be attributed to the multifaceted action mechanism of silver and its distinctive surface distribution. Unlike CHX, which exerts a single-target effect, Ag⁺ ions interact with thiol (–SH) groups in microbial cell wall proteins, disrupt membrane integrity, and induce the generation of ROS, leading to protein denaturation and DNA damage [82–84]. The larger inhibition zones observed for ES@Ag⁺ compared to the calcined C-ES@Ag⁺ sample against both E. coli and C. albicans can be explained by differences in surface chemistry. The presence of carbonate groups in the non-calcined matrix promotes the localized accumulation of Ag⁺ ions, resulting in the formation of Ag rich “active sites,” as supported by EDX analyses, thereby inducing a stronger antimicrobial response. In contrast, the more homogeneous but diluted distribution of silver on the calcined surface limits the release rate of Ag⁺ ions, leading to relatively reduced activity. Consequently, although C-ES@Ag⁺ demonstrates antimicrobial potential in diffusion-based assays, its reduced ion release under dynamic conditions results in lower activity in MIC evaluations. Overall, these results highlight that Ag⁺-loaded eggshell-based nanocomposites, particularly ES@Ag⁺, exhibit strong and consistent antimicrobial performance across both solid and liquid assay systems, suggesting their potential as sustainable and effective alternatives to conventional antimicrobial agents such as CHX. Recent studies (2023–2026) have explored a wide range of silver-based antimicrobial platforms, from synthetic polymers to advanced nano-frameworks [85–89]. In this broader context, our eggshell-derived nanocomposite offers a highly sustainable and cost-effective alternative, achieving comparable antimicrobial performance through a green synthesis route.

This study highlights the critical role of the native eggshell structure in antimicrobial performance. While calcination introduces an additional processing step and alters the material, the non-calcined porous CaCO₃ matrix provides an effective platform for Ag⁺ loading and controlled release. This structural advantage enables the ES@Ag⁺ system to exhibit consistent antimicrobial activity in both solid and liquid media. Therefore, avoiding calcination not only simplifies the synthesis process but also enhances the functional efficiency of the material.

Conclusion

This work provides compelling evidence that waste eggshells can be transformed into highly functional Ag⁺ based nanocomposites without the formation of metallic silver nanoparticles, offering a more sustainable alternative to conventional AgNP systems. By selectively exploiting the intrinsic chemistry of CaCO₃ and CaO based matrices, this study demonstrates that the immobilization mode of Ag⁺ ions critically governs both the photophysical and antimicrobial behavior of the material. The absence of crystalline silver reflections in XRD patterns confirms that silver is stabilized in ionic or amorphous surface-bound states, rather than as metallic nanophases. Notably, the pronounced green photoluminescence observed in the non-calcined ES@Ag⁺ sample (480–520 nm) originates from Ag⁺ induced radiative defect centers formed through strong interactions with carbonate groups and residual membrane derived functional moieties, establishing a direct link between surface chemistry and optical response. This same localized Ag⁺ enrichment gives rise to highly active antimicrobial sites, enabling ES@Ag⁺ to exhibit antibacterial and antifungal performance against Escherichia coli and Candida albicans that approaches that of chlorhexidine, a widely used clinical antiseptic. In contrast to the rapid and aggressive membrane disruption caused by chlorhexidine, the Ag⁺ modified eggshell system offers a more controlled and surface mediated antimicrobial action, which is advantageous in terms of long term efficacy and biocompatibility. Furthermore, the comparative analysis between calcined and non-calcined matrices reveals that preserving the carbonate rich structure is critical for maximizing Ag⁺ localization and biological performance, whereas calcination leads to a more dispersed ion distribution and attenuated activity. While these findings strongly suggest the presence of Ag⁺ species, future studies involving XPS and time-resolved photoluminescence decay measurements are recommended to precisely elucidate silver oxidation states and electronic transition mechanisms. Furthermore, as the lack of cytotoxicity and biocompatibility data remains a limitation, systematic in vitro and in vivo investigations are necessary to ensure biosafety for potential biomedical applications. These subsequent evaluations will be critical to confirm the safe and effective implementation of these eggshell-derived nanocomposites. Collectively, these findings position Ag⁺ functionalized eggshells as a unique waste-derived platform in which optical signatures, surface defect chemistry, and antimicrobial functionality are intrinsically coupled, opening new opportunities for low-cost biomedical materials, wound care technologies, and sustainable disinfection strategies within a circular economy framework.

Author contributions

Büşra Şensoy Gün: Methodology, Investigation, Supervision, Resources, Conceptualization, Data curation, Writing—original draft, Writing—Review & Editing.

Funding

The author(s) received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participates

Not applicable. This study does not involve human participants or animals.

Consent for publications

Not applicable. This manuscript does not contain any individual person’s data in any form.

Competing interests

The authors declare no competing interests.