Molten salt-assisted one-pot synthesis of Ag nanoparticles supported on clay minerals for enhanced antibacterial performance

Abstract

Supported silver nanoparticles (AgNPs) have been extensively used as antibacterial agents in biomedicine, biotechnology, and environmental remediation. However, a facile and scalable method for preparing homogeneously dispersed AgNPs on clay minerals remains a challenge. In this study, a one-pot method was successfully developed for the synthesis of homogeneously dispersed AgNPs supported on the surface of clay minerals (e.g., montmorillonite (Mt) and palygorskite (Pal)). Typically, clay minerals were mixed with AgNO₃ (as a precursor) and NaNO₃ (as a dispersant) by thorough grinding in a mortar, and then the mixture was heated slowly. AgNO₃ undergoes thermal decomposition to generate AgNPs via a self-reduction process, without the assistance of any external reductants. The free ions dissociated by molten NaNO₃ inhibit the aggregation of AgNPs. Specifically, AgNPs were uniformly dispersed on Mt and Pal. Correspondingly, the average particle sizes of the AgNPs were determined to be 10.71 ± 2.16 nm for 6% Ag/Mt-s and 6.07 ± 3.26 nm for 6% Ag/Pal-s, respectively. The antibacterial performance of the nanocomposites was associated with both the concentration of the target materials and their stability in the medium. Specifically, the physicochemical properties of Pal facilitated the small particle size of AgNPs, which in turn enhanced their antibacterial activity. The findings of this study highlight the advantages of utilizing clay minerals as supports to realize the high antibacterial activity of AgNPs. Meanwhile, this study provided a novel and facile strategy for synthesizing silver/clay mineral nanocomposites with homogeneously dispersed silver nanoparticles supported on the surface of clay minerals, without chemical reductants or surfactants. This study provided a theoretical basis for the design and preparation of high-efficiency, low-cost antibacterial silver/clay mineral nanocomposites in the future.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-37682-w.

Introduction

Multidrug-resistant (MDR) bacteria represent a pivotal challenge to global public health and are listed among the top ten health threats by the World Health Organization (WHO)^1,2. By 2050, annual deaths from drug-resistant bacterial infections worldwide are expected to surge beyond 10 million³. Amid this crisis, the current antimicrobial therapy field faces three key issues: depleted natural antimicrobial resources⁴, potential toxic side effects of synthetic antimicrobial drugs⁵, and the rapid evolution of drug-resistant strains⁶. Therefore, developing novel antimicrobial materials with broad-spectrum activity and high antibacterial activity has become a vital research focus.

Metallic nanoparticles (e.g., silver, gold, platinum) have garnered considerable attention in the antibacterial materials field, owing to their multiple bactericidal mechanisms and facile synthesis^7,8. Their unique physicochemical properties, including nanoscale size, high specific surface area, and controllable dimensionality, endow them with efficient, targeted, and broad-spectrum antibacterial activity⁹. Among these, silver nanoparticles (AgNPs) stand out as promising alternatives, given their environmental friendliness, compatibility with large-scale production, and broad-spectrum antimicrobial efficacy against fungi, bacteria, and viruses¹⁰. The antibacterial mechanisms of AgNPs mainly involve the following pathways: AgNPs can penetrate microbial cells to disrupt membranes^11,12 induce oxidative stress via reactive oxygen species (ROS) generation^13,14, and further cause DNA damage^15,16. Specifically, using the H₂DCFDA fluorescence probe assay, Choi & Hu (2008) quantitatively detected a marked elevation of intracellular ROS in AgNPs-treated nitrifying cultures, confirming that AgNPs induce intracellular ROS generation and accumulation, with the associated antibacterial inhibition strongly correlating with intracellular ROS concentrations¹⁷. However, the practical application of AgNPs is constrained by inherent limitations, such as nonspecific toxicity to mammalian cells, easy agglomeration, and poor stability.

To overcome these challenges, numerous studies have focused on developing silver-based composite antibacterial materials. The introduction of carriers (e.g., carbon-based nanomaterials, metal/non-metal oxides, and metal–organic frameworks) enables AgNPs size regulation, antibacterial performance optimization, and biological toxicity reduction. For instance, Nicosia et al. loaded uniformly dispersed AgNPs (5–12 nm average size) onto graphene-polyvinyl alcohol (PVA) covalent composites. The carrier suppressed AgNPs agglomeration via steric hindrance, and the composite showed far better antibacterial activity than pure AgNPs¹⁸. Shuai et al. immobilized AgNPs on dopamine-modified mesoporous bioactive glass, utilizing mesoporous channels for physical confinement¹⁹. This composite exerts antibacterial effects through following mechanisms: released Ag⁺ electrostatically bonds to sulfhydryl groups (–SH) groups of bacterial membrane proteins, disrupting membrane permeability. Meanwhile, both AgNPs and Ag⁺ induce ROS (e.g., ·OH, O₂⁻, and H₂O₂) generation, which damages DNA, inhibits mRNA replication and transcription, and ultimately leads to bacterial death.

Despite the excellent performance of the aforementioned carriers, their practical applications are limited by high preparation costs, complex processes, and toxic reagent involvement. Clay minerals (e.g., montmorillonite, palygorskite, and halloysite) are suitable for large-scale production due to their abundant reserves, environmental friendliness, and low cost^20–23. Moreover, their surface defects and porous channels can induce heterogeneous nucleation of AgNPs, achieve precise size control by limiting growth space, and effectively prevent agglomeration^24,25. Furthermore, the permanent structural charge of clay minerals boosts bacteria-material contact efficiency via electrostatic adsorption (inducing physical damage to bacterial membranes), while nanosheet-structured clay minerals regulate sustained Ag⁺ release, reducing toxicity from instantaneous high concentrations and improving biocompatibility^26,27. Thus, clay minerals serve as effective carriers to inhibit agglomeration of AgNPs, control their particle size, and enhance antibacterial activity of composites²⁸.

Clay minerals of different structural types vary greatly in properties (including size distribution, specific surface area, and surface charge), which significantly affect the size, dispersion, surface properties and antibacterial performance of AgNPs. Montmorillonite (Mt) with lamellar structure, possesses high cation exchange capacity and a modifiable interlayer space, allowing uniform AgNPs loading via ion exchange or intercalation and regulating Ag⁺ release, resulting in excellent antibacterial effects^26,29. Palygorskite (Pal) with fibrous structures bonds AgNPs via surface hydroxyls and immobilizes AgNPs by abundant pores, leading to improved dispersion of AgNPs and sustained antibacterial activity^30,31. Additionally, clay surface charges (e.g., Mt with negative surface charge) further regulate antibacterial performance by affecting the dispersion of AgNPs and bacterial adsorption^32,33. Therefore, investigating the regulatory mechanisms of clay minerals with different structures on the antibacterial performance of AgNPs is of great significance for the design of high-efficiency antibacterial materials.

In this study, a green and efficient one-pot synthesis strategy was developed to achieve the uniform dispersion of AgNPs on the surface of clay minerals (montmorillonite and palygorskite). Structural characterization of the silver nanoparticle/clay mineral nanocomposites was performed using X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy. In vitro antibacterial activity tests were conducted against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. This study aims to reveal how the properties of clay minerals affect the antibacterial performance of silver/clay mineral nanocomposites.

Materials and methods

Materials

The raw montmorillonite (Mt) was calcium-rich (Ca-Mt, purity > 95%) and collected from Inner Mongolia. Raw palygorskite (Pal) was obtained from Xuji, Jiangsu Province, China. Sodium nitrate (NaNO₃, 99%) and silver nitrate (AgNO₃, ACS reagent, 99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Luria–Bertani (LB) medium powder, Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria Staphylococcus aureus strain (e.g., ATCC 25923, S. aureus) were all purchased from Guangzhou Huankai Microbial Technology Co., Ltd., (Guangzhou, China). All the chemical agents were of analytical grade and used as received.

Preparation of silver/clay mineral nanocomposites

Preparation of silver/montmorillonite nanocomposite

The silver/montmorillonite nanocomposites were fabricated via a one-pot method (Fig. 1). Briefly, 1 g of Ca-Mt, 0.142 g of AgNO₃ and 0.5 g of NaNO₃ were mixed and thoroughly ground in a mortar for 30 min. The resulting mixture was transferred to a quartz boat, then heated to 310 °C at a ramp rate of 2 °C min⁻¹ and held at this temperature for 2 h, and argon was introduced throughout the heating process as an inert protective gas. After cooling to room temperature, the mixture was repeatedly washed with deionized water and centrifuged to remove residual NaNO₃. Subsequently, the silver/montmorillonite nanocomposite was obtained after freeze-drying for 24 h. This product was denoted as 6%Ag/Mt-s, where the 6% refers to the mass percentage of Ag. For comparison, 6%Ag/Mt-w sample was synthesized using the same procedure but without the addition of NaNO₃.

Fig. 1.

Fabrication strategy of silver/clay mineral nanocomposites.

Preparation of silver/palygorskite nanocomposite

The silver/palygorskite nanocomposite was prepared using the same method as the silver/montmorillonite nanocomposite described above. The resulting product was designated as 6%Ag/Pal-s.

Characterization

The crystalline properties of the synthesized nanocomposite materials were characterized by X-ray diffraction (XRD) using CuKα radiation monochromatized with graphite on a Bruker D8 Advance X-ray diffractometer. Transmission electron microscopy (TEM) micrographs and high-resolution TEM (HRTEM) images were acquired using an FEI Talos F200S field-emission transmission electron microscope (sourced from Brno, Czech Republic), operating at an acceleration voltage of 200 kV. To determine the crystal lattice parameters, Fast Fourier Transform (FFT) analysis was performed with the aid of TEM Image & Analysis (TIA) software. Meanwhile, the measurement and statistical analysis of Ag particle dimensions were carried out using ImageJ software (Version 1.54d, https://imagej.net/ij/index.html). X-ray photoelectron spectroscopy (XPS) measurements were conducted using a Thermo Fisher K-Alpha XPS system (manufactured in Waltham, Massachusetts, USA). For sample preparation prior to analysis, the powdered material was pressed onto double-sided adhesive tape to form compact pellets, which were then subjected to XPS testing. To correct for spectral shifts, the C 1 s peak (with a binding energy of 284.8 eV) originating from accidental carbon-based contaminants on the sample surface was employed as the reference standard. The Ag element analysis was conducted using an Agilent 5110 inductively coupled plasma optical emission spectroscopy (ICP-MS) instrument (manufactured in Santa Clara, California, USA).

Evaluation of Ag ion‑release behavior

The release concentration of Ag⁺ from the nanocomposites was monitored in deionized water. A quantity of 20 mg of 6%Ag/Mt-s and 6%Ag/Pal-s was transferred into a dialysis bag (molecular weight cutoff: 1000 Da). Then, the dialysis bag was immersed in 100 mL of deionized water at 37 °C and continuously stirred at 200 rpm. After various time intervals (0, 2, 4, 6, 8, 10, and 12 h), 5 mL of dialysis solution was collected to determine the concentration of released Ag⁺. Subsequently, each aliquot of the dialysis solution (1 mL) was mixed thoroughly with 1.0 mL of 10% (m/v) nitric acid (HNO₃), and the resulting mixture was magnetically stirred at room temperature for 24 h, and the total Ag⁺ concentration was quantified using an Agilent 5110 ICP-OES instrument.

Antibacterial activity of silver/clay mineral nanocomposites

Cultivation of bacteria

E. coli and S. aureus were transferred from LB solid plates to conical flasks containing fresh LB liquid medium and subsequently cultured in an incubator at 37 °C and 200 rpm. After being cultured for 4 to 5 h, the bacterial suspensions were serially diluted tenfold to 10⁶ CFU/mL.

Antibacterial kinetic study

20 μL of bacterial suspension was added to 380 μL of LB liquid medium containing varying concentrations of 6%Ag/Mt-s and 6%Ag/Pal-s (0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) in a 96-well plate. Then, the plate was shaken at 37 °C, 200 r/min. After various time intervals (0, 2, 4, 6, 8, 10, and 12 h), the optical density at 600 nm (OD600) of the bacterial suspension was measured using a microplate reader to dynamically monitor bacterial growth over time. All experiments were conducted in duplicate to guarantee the reproducibility of the results.

Colony number experiment

The minimum bactericidal concentration (MBC) of 6%Ag/Mt-s and 6%Ag/Pal-s against E. coli and S. aureus was determined using the colony counting method. 0.5 mL of the bacterial suspension (10⁶ CFU/mL) was introduced into LB liquid medium within sterile colorimetric tubes containing varying concentrations (0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 mg/mL) of 6%Ag/Mt-s and 6%Ag/Pal-s. The mixtures were then incubated in a shaking incubator at 37 °C for 6 h. Following incubation, the microbial suspensions were diluted 10⁵ times, and 50 μL of each diluted suspension was spread onto solid LB agar medium and incubated at 37 °C for 12 h. Bacterial viability was assessed by counting the resulting colony-forming units. All experiments were conducted in duplicate to guarantee the reproducibility of the results.

Results and discussion

XRD patterns of synthesized silver/clay minerals.

The XRD patterns of raw Mt and Pal, as well as silver/clay mineral nanocomposites (i.e., 6%Ag/Mt-s and 6%Ag/Pal-s, Fig. 2), were presented respectively. Ca-Mt exhibited a d₀₀₁-value of 1.51 nm, which corresponds to the presence of Ca²⁺ with two hydration layers within its interlayer spaces³⁴. The XRD pattern of the synthesized 6%Ag/Mt-s nanocomposite revealed that the characteristic reflections of Mt are presented, suggesting that the Mt structure was not destroyed during the heating process (Fig. 2a). The 2θ values of 38.1°, 44.2° and 77.4° correspond to the (111), (200) and (311) crystal planes of cubic Ag, confirming the formation of crystalline Ag with the Fm^—3 m (225) space group (JCPDS 87–0717)³⁵. This indicates that AgNO₃ undergoes thermal decomposition to generate AgNPs via a self-reduction process, without the assistance of any external reductants. This self-reduction process could be attributed to the high reduction potential of Ag⁺/Ag (+ 0.80 V)³⁶. The formation of AgNPs in silver-exchanged Mt without the use of a reducing agent was also reported by Costa et al.³⁷. Notably, the d-spacing of 6%Ag/Mt-s remained unchanged compared with pristine Ca-Mt, demonstrating that AgNPs were not intercalated into the interlayer structure of Mt. As anticipated, no diffraction peaks corresponding to AgO or AgNO₃ were detected, indicating the complete decomposition of AgNO₃ and the absence of AgO by-product formation³⁸.

Fig. 2.

(a) XRD patterns of Ca-Mt and 6%Ag/Mt-s, and (b) Pal and 6%Ag/Mt-s; (c and d) XPS survey spectra and high-resolution Ag 3d XPS spectra of 6% Ag/Mt-s, and (e and f) XPS survey spectra and high-resolution Ag 3d XPS spectra of 6% Ag/Pal-s.

Pal showed diffraction peaks at 8.40°, 13.71°, 16.36°, 19.89°, 21.48°, and 24.25° 2θ, which corresponded to the (110), (200), (130), (040), (300), and (240) planes (Fig. 2b)³⁸. Similarly, in the XRD pattern of the 6%Ag/Pal-s nanocomposite, characteristic diffraction peaks of metallic Ag⁰ were observed at 2θ values of 38.17°, 44.2°, 64.5°, and 77.4°, confirming the successful synthesis of metallic silver. In contrast, the diffraction peak of Pal at 8.42° (corresponding to the (110) crystal plane) showed no obvious shift or intensity reduction, indicating that the crystal structure remained intact. Notably, the Ag loading concentration also exerted a significant influence on the antibacterial activity of the silver/clay mineral nanocomposites (Fig. S1), with the optimal performance achieved at a silver loading amount of 6%.

Characterization by XPS

The XPS characterization was performed to investigate the chemical states of elements and composition in 6%Ag/Mt-s (Fig. 2c and 2d) and 6%Ag/Pal-s (Fig. 2e and 2f). The signal peaks at 74.9 eV, 103 eV, and 1303 eV were assigned to the Al 2p, Si 2p, and Mg 1 s orbitals of 6%Ag/Mt-s (Fig. 2c) and 6%Ag/Pal-s (Fig. 2e), respectively. The high-resolution Ag 3d XPS spectra of 6%Ag/Mt-s (Fig. 2d) and 6%Ag/Pal-s (Fig. 2f) could each be deconvoluted into two dominant peaks, with the four total peaks centered at approximately 368.3, 369.4, 374.2, and 375.4 eV (Fig. 2d and 2f). The binding energies at 368.3 and 374.2 eV corresponded to metallic Ag^39,40, indicating the successful formation of AgNPs. The binding energies observed at approximately 369.4 and 375.4 eV corresponded to Ag⁺, which could be attributed to the Ag⁺ generated during the surface oxidation of metallic Ag. The XPS results further confirmed the presence of metallic Ag in 6%Ag/Mt-s and 6%Ag/Pal-s, which is consistent with the previously obtained XRD results.

Characterization by TEM

The morphologies of 6%Ag/Mt-s and 6%Ag/Pal-s were characterized via TEM (Fig. 3). For the 6%Ag/Mt-s sample, TEM images revealed the sheet-like structure of Mt (Fig. 3a), which enables Ag⁺ to enter its interlayer spaces via a cation exchange process. A large number of small and spherical AgNPs with an average size of 10.71 ± 2.16 nm (Fig. S2) were uniformly decorated on the external surface of Mt layers (Fig. 3a and 3b), which is consistent with the findings reported by Gil-Korilis et al.²⁶. The surface of Mt served as a physically stable substrate to promote the heterogeneous nucleation for AgNPs³⁶. The good dispersion of AgNPs in 6%Ag/Mt-s was confirmed by the homogeneous bright spots in the dark-field TEM image (Fig. 3k). Further evidence for the well-defined crystalline structure of Ag was provided by the selected area electron diffraction (SAED) pattern of 6%Ag/Mt-s (Fig. 3e). In contrast, when NaNO₃ was omitted from the synthesis process, significant agglomeration of AgNPs was observed in the 6% Ag/Mt sample (Fig. S3). This phenomenon could be attributed to the following reasons. NaNO₃ was generally introduced as a heat scavenger to absorb the heat via the endothermic effect of molten salts⁴¹, thereby effectively suppressing the growth of the AgNPs. Additionally, NaNO₃ dissociated into abundant free Na⁺ and NO₃⁻ ions. Among these, NO₃⁻ ions exhibit strong affinity for AgNPs, and their adsorption significantly increases surface negative charge of the particles (Fig. S4), which inhibits the aggregation of AgNPs via the electrostatic shielding effect^42,43.

Fig. 3.

TEM images of (a and b) 6% Ag/Mt-s and (f and g) 6% Ag/Pal-s; HRTEM, STEM images, and element mapping images (scale bar, 50 nm) of (c and k) Ag/Mt-s and (h and l) 6% Ag/Pal-s.

For the 6%Ag/Pal-s sample, the high-magnification TEM images shown (Fig. 3g) revealed the nanorod morphology of Pal. AgNPs were highly homogeneously distributed on the external surface of Pal, which could be attributed to negative surface charge of Pal. The TEM images (Fig. 3f and g) showed that AgNPs with an average size of 6.07 ± 3.26 nm (Fig. S2) were uniformly decorated on Pal nanorods, which is smaller than that of AgNPs on Mt (10.71 ± 2.16 nm). This result could be attributed to the large specific surface area (Table. S1), regular surface channels of Pal nanorods, and their abundant negatively charged surface-active groups (Si–OH, Al–OH, Si–O⁻, and Al-O⁻ groups)³⁸. These unique properties endow Pal nanorods with the ability to effectively bond Ag⁺ ions (via hydrogen bonds or electrostatic interactions) to form Ag⁺-O interactions, adsorb Ag⁺ ions, and efficiently immobilize AgNPs^30,35,44,45. The SAED pattern of the 6%Ag/Pal-s sample confirmed that the Ag exhibited a well-defined crystalline structure (Fig. 3j). To further identify the chemical composition of the two nanocomposites, corresponding energy dispersive X-ray spectroscopy element mapping analyses were carried out. As shown in Fig. 3k (for 6%Ag/Mt-s) and Fig. 3l (for 6%Ag/Pal-s), the element mapping images revealed that the distributions of aluminum (Al, green), silicon (Si, yellow), and oxygen (O, orange) were uniformly distributed in the nanocomposites. Meanwhile, the presence of silver (Ag, blue) certified that AgNPs were successfully and uniformly dispersed on Mt and Pal. Overall, both 6%Ag/Mt-s and 6%Ag/Pal-s nanocomposites exhibit good homogeneity.

Release of silver ions from nanocomposites

To investigate the Ag⁺ release behavior of the nanocomposites, the cumulative release of Ag⁺ from 6%Ag/Mt-s and 6%Ag/Pal-s was monitored over 2, 4, 6, 8, 10, and 12 h in distilled water (DW). A difference in the behavior of Ag⁺ release was observed as a function of the clay minerals (Mt or Pal) used in the nanocomposites (Fig. 4). The immobilization of AgNPs on clay minerals substrates reduced agglomeration and increased the contact surface with DW, thus facilitating sustained Ag⁺ release over time. Generally, 6%Ag/Pal-s exhibited a much faster release behavior and the cumulative release concentration of Ag⁺ reached ~ 0.6 µg mL⁻¹ at 12 h, which is approximately 8 times that of 6%Ag/Mt-s (~ 0.08 µg mL⁻¹). This result could be attributed to the structural characteristics of Pal and Mt, which modulated Ag⁺ release. For 6%Ag/Pal-s, the distribution of AgNPs on the surface of Pal, together with the structural stability and surface channels of Pal, boosted the release of Ag⁺ through the Pal matrix to the external surface. In contrast, the layered structure of Mt induced AgNPs intercalation, which ultimately caused AgNPs oxidation and reduced dissolution of Ag^{+ 46}.

Fig. 4.

Cumulative release profiles of Ag ions from 6%Ag/Mt-s and 6%Ag/Pal-s at 37 °C.

The release pattern of Ag⁺ from 6%Ag/Mt-s and 6%Ag/Pal-s indicated that the release process correlated with the exposed surface area and distribution of AgNPs in the nanocomposites⁴⁷. During the first 2 h of 6%Ag/Pal-s in DW, an “ionic burst” of Ag⁺ occurred, with more than 25% of the total Ag⁺ released. This initial fast release could be attributed to the corrosion and dissolution of AgNPs that were most exposed to DW, such as those located on the edges of Pal. However, from 2 to 10 h of contact, a continuous slow release of Ag⁺ was observed. This stage was related to the release of Ag⁺ from AgNPs located inside the Pal structure or pores, due to the limited access of these AgNPs to the external environment, resulting in slow Ag⁺ release. Based on the above results, the cumulative Ag⁺ release of 6%Ag/Pal-s was higher than that of 6%Ag/Mt-s within a specified time (e.g., 12 h).

Analysis of antibacterial experiment

The physicochemical properties of Mt and Pal significantly influence the release behavior of AgNPs and their interaction with bacteria, leading to differences in the antibacterial activities of silver/clay mineral nanocomposites. Two representative multi-drug-resistant bacterial strains, Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus, were selected to evaluate the antibacterial activity of silver/clay mineral nanocomposites. The growth kinetics experiments on 6%Ag/Mt-s and 6%Ag/Pal-s against both bacterial strains were conducted to demonstrate antibacterial performance (Fig. 5). The bacteria growth was monitored over 12 h at different time points by measuring the optical density at 600 nm (OD600). As shown in Fig. 5a, the 6%Ag/Mt-s sample could completely inhibit the growth of E. coli up to 12 h at 0.8 mg mL⁻¹. It is noteworthy that the proliferation of E. coli was still inhibited within the first 4 h, even when the concentration of 6%Ag/Mt-s was reduced to 0.6 mg mL⁻¹. For 6%Ag/Pal-s, a concentration of 0.4 mg mL⁻¹ could effectively reach a bacterial growth inhibition rate of 99% (Fig. 5c). Besides, a similar trend was found for S. aureus groups (Fig. 5d), which is consistent with the above findings of E. coli. However, the 6%Ag/Mt-s sample could not completely inhibit the growth of S. aureus, even with a concentration of 1.0 mg mL⁻¹. Previous studies have demonstrated that the interaction between AgNPs and Mt increases the positive specific surface charges^20,48,49, thereby reducing the adsorption capacity for S. aureus, which possesses positively charged cell walls. This phenomenon could be attributed to the reduced electrostatic adsorption of 6%Ag/Mt-s toward S. aureus. The above results suggested that 6%Ag/Pal-s possesses significantly stronger antibacterial properties than 6%Ag/Mt-s against multi-drug-resistant bacteria. These results could be attributed to the AgNPs loaded on Pal, which exhibit a smaller particle size (~ 6.07 nm, compared with those on Mt), higher efficiency in Ag⁺ release, and an increased probability of bacterial contact per unit volume. This thereby facilitates AgNPs penetration through cell membranes and inhibits bacterial growth by interfering with the replication of genetic material (RNA).

Fig. 5.

Effects of nanocomposites (a and b) 6%Ag/Mt-s and (c and d) 6%Ag/Pal-s on the growth of E. coli and S. aureus over 12 h at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL⁻¹.

The minimum bactericidal concentration (MBC) of 6%Ag/Mt-s and 6%Ag/Pal-s against E. coli and S. aureus was determined using the colony counting method (Fig. 6). The results showed that pristine clay minerals (Mt and Pal) and NaNO₃-treated clay minerals (Mt-s and Pal-s) exhibited only weak antibacterial activity (Fig. S5), compared with the positive control, both 6%Ag/Mt-s and 6%Ag/Pal-s showed excellent antibacterial activities against E. coli, and the antibacterial activities increased regularly with the increase of sample concentration with the minimum bactericidal concentration of 0.2 mg/mL and 0.6 mg/mL, respectively. In contrast, 6%Ag/Mt-s and 6%Ag/Pal-s were less effective against S. aureus compared to E. coli. The antibacterial rate of 6%Ag/Pal-s reached 99.9% with the sample concentration of 0.8 mg/mL. Although 6%Ag/Mt-s did not completely kill S. aureus, the number of colonies was obviously less than that of positive control, which indicated that 6%Ag/Mt-s had certain inhibitory effect on S. aureus. The above results suggested that the antibacterial activity of 6%Ag/Mt-s is weaker than that of 6%Ag/Pal-s, which could be attributed to the small particle size of AgNPs in 6%Ag/Pal-s. Generally, the size of nanoparticles plays a key role in the enhancement of antibacterial activity^50,51. In conjunction with the TEM results, the AgNPs loaded on Pal with small particle size enable higher antibacterial activity due to their strong membrane permeability and large specific surface area. Additionally, Pal can also abrade bacterial cell walls, thereby impacting cell wall permeability³⁵. These effects enhance the antibacterial effect of 6%Ag/Pal-s nanocomposite. Consequently, Pal serves as the optimal carrier for the synthesis of silver/clay mineral nanocomposites.

Fig. 6.

Antibacterial activities of 6%Ag/Mt-s and 6%Ag/Pal-s. (a and c) The agar plate photographs of bacterial colonies obtained by E. coli and S. aureus after exposure to different materials; The Survival analysis of 6%Ag/Mt-s and 6%Ag/Pal-s against (b) E. coli and (d) S. aureus.

As mentioned above, the silver/clay mineral nanocomposites prepared in this study exhibited enhanced antibacterial activity against both multi-drug-resistant bacteria E. coli and S. aureus. Several key mechanisms underlying this enhanced activity can be summarized as follows. First, clay minerals carriers (e.g., Mt and Pal) play a critical role in stabilizing AgNPs and regulating Ag⁺ release. Specifically, they prevented the aggregation of loaded AgNPs and enabled the sustained release of Ag⁺ (Fig. 3). The released Ag⁺ exerted antibacterial effects through two main pathways: Ag ions primarily interacted with the sulfhydryl groups (–SH) of bacterial proteins, leading to the inactivation of enzymatic proteins and the disruption of essential metabolic processes⁵²; Ag ions significantly elevated the levels of ROS in bacterial cells. Excessive ROS inhibits bacterial respiration, induces DNA damage, and ultimately results in bacterial cell death^15,19. Second, the unique structural features of clay minerals contribute to the size control of AgNPs, which further enhances antibacterial activity. The porous structure of Pal and the interlayer space of Mt act as nanoreactors to confine AgNPs growth, thereby reducing their particle size. As previously established, small-size AgNPs exhibit higher antibacterial activity, which is attributed to their faster Ag⁺ release rate and stronger ability to penetrate microbial cell membranes. Consistent with this mechanism, the average diameter of AgNPs loaded on Pal in this study was 6.07 ± 3.26 nm, which was significantly smaller than that on Mt (10.71 ± 2.16 nm). Correspondingly, the antibacterial activity of 6% Ag/Pal-s was higher than that of 6% Ag/Mt-s (Figs. 4 and 5). Third, the enhanced antibacterial activity of 6% Ag/Pal-s might also stem from the synergistic effect between AgNPs and the Pal carrier. On one hand, Pal could cause physical damage to bacterial cell membranes, which not only imparts moderate intrinsic antimicrobial activity but also creates channels for AgNPs or Ag⁺ ^53,54. On the other hand, this membrane damage facilitated the penetration of AgNPs/Ag⁺ into the bacterial cytoplasmic matrix, where they interact with intracellular components (e.g., DNA, proteins, and lipids) and caused irreversible damage, further reinforcing the antibacterial effect.

Conclusion

In conclusion, homogeneously dispersed AgNPs supported on the surface of clay minerals were successfully fabricated via a simple and eco-friendly one-pot method. The as-synthesized silver/clay mineral nanocomposite exhibited high antibacterial performance, which is closely correlated with the structural characteristics and surface properties of the clay minerals. During synthesis, AgNO₃ generated silver nanoparticles through a self-reduction process, while free ions dissociated from molten NaNO₃ inhibited the aggregation of silver nanoparticles. Structural characterizations confirmed that the small-sized, uniformly dispersed silver nanoparticles were loaded onto the surface of montmorillonite (10.71 ± 2.16 nm) and palygorskite nanorods (6.07 ± 3.26 nm), respectively. These results indicated that the introduction of montmorillonite and palygorskite significantly reduces the particle size of silver nanoparticles. Correspondingly, the prepared silver/palygorskite nanocomposite exhibited better antibacterial activity against E. coli and S. aureus than the silver/montmorillonite nanocomposite. In summary, this work elucidated the structure-performance relationship between clay mineral and silver/clay mineral nanocomposites and provided a theoretical basis for the design and preparation of high-efficiency, low-cost antibacterial silver/clay mineral nanocomposites in the future.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Q.Y., G.K , and Y.X:Investigation, Data curation, Q.Z: Writing—original draft, Project administration, Funding acquisition, Writing—review and editing, Q.Y: Resources, Z.J: Funding acquisition. All authors reviewed the manuscript.

Funding

Funding sources are listed in the Acknowledgments.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.