Functionalized Iron Oxide-Silver Nanohybrids for Enhanced Germination of Sorghum

Functionalized Iron Oxide-Silver Nanohybrids for Enhanced Germination of Sorghum Martina Mercurio, ○ Adriano Patriarca, ○ Sara Cerra,*Farid Hajareh Haghighi, Fabio Sciubba, Emma Cocco, Giulia Giorgi, Francesco Mura, Alessio Talone, Roberto Matassa, Juan G. Lozano, Ida Pettiti, Pier Giorgio Schiavi, Maria Pia Donzello, Andrea Angelucci, Ilaria Fratoddi, and Elisa Brasili* Cite This: ACS Appl. Nano Mater. 2025, 8, 9227−9242 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: In this work, hydrophilic FeOx-AgNPs nanohybrids were obtained by covalent interaction between functionalized maghemite nanoparticles (γ-Fe2O3NPs) and silver nanoparticles (AgNPs). An in situ chemical reduction of the AgNO3precursor was carried out in the presence of sodium 3-mercapto-1propanesulfonate (3MPS) stabilizer to further improve colloidal stability and aqueous dispersibility of the nanohybrids. The bifunctional linker (3-mercaptopropyl)trimethoxysilane (MPTMS) enabled the γ-Fe2O3NPs surface coating through Fe− O−Si chemical bonds, exposing the −SH ending moiety for covalent decoration of AgNPs-3MPS through covalent Ag−S bonds. Two nanohybrids, namely, FeOx-AgNPs_1 and FeOxAgNPs_10, were obtained by tuning the Fe/Ag weight ratio, resulting in Ag content of 4−15 wt %, as confirmed by inductively coupled plasma optical emission spectrometry (ICP-OES) measurements. The deep characterizations by UV−Vis spectroscopy, dynamic light scattering (DLS) and ζ-potential, farand midFourier-transform infrared (FTIR) and Raman spectroscopy, high-resolution transmission electron microscopy (HR-TEM), powder X-ray diffraction (PXRD), elemental analysis, and magnetic susceptibility measurements confirmed their colloidal nature, aqueous stability, surface functionalization, and ferromagnetic behavior. Both hybrids were tested as nanoprimers (0.01−100 ppm concentration range) on Sorghum bicolor (L.) Moench Bianca seeds. FeOx-AgNPs_1, containing the lower Ag content, significantly improved the germination rate without phytotoxic effects at lower concentrations (0.01−1 ppm, i.e., 0.0015−0.15 ppm of metallic Ag). 1H NMR metabolomic analysis revealed a dose-dependent regulation of amino acid and carbohydrate content, indicating an osmotic adjustment mechanism. The results of this research highlight the possibility of combining γ-Fe2O3NPs-MPTMS with AgNPs-3MPS in a single covalent hybrid nanostructure, demonstrating their potential within the frame of nanoprimers as a sustainable alternative to conventional agrochemical treatments. KEYWORDS: iron oxide nanoparticles, functionalized silver nanoparticles, hydrophilic nanohybrids, Sorghum bicolor (L.) Moench Bianca, germination, characterization, 1H-NMR based metabolomics 1. INTRODUCTION In recent years, metal oxide nanoparticles, such as iron oxide (IONPs, i.e., magnetite Fe3O4or maghemite Fe2O3), silver nanoparticles (AgNPs), and nanohybrids, have attracted great interest due to their doseand surface functionality-dependent biocompatibility, chemical stability, low cost, and peculiar optical, antibacterial and magnetic properties. 1,2 These features make them valuable in a variety of fields, including biomedicine, environmental remediation, and agriculture, among others. 1−3 Conventional agricultural practices and agrochemicals are approaching their limits to boost agricultural production, adversely affecting the sustainability of the environment, and in this field, nanomaterials have the potential to revolutionize agricultural systems. Pesticide or fertilizer formulations show limitations such as high solvent content, dust drift, poor dispersion, and soil persistence, 4 with only 1% reaching target surfaces, leading to environmental contamination. Sorghum bicolor (L.) Moench grain is a staple food for populations in rural and resource-poor regions, ranking as the fifth most important cereal in the world. Harvested sorghum is used primarily for feed and ethanol production and as a food Received: February 4, 2025 Revised: April 10, 2025 Accepted: April 11, 2025 Published: April 29, 2025 Articlewww.acsanm.org © 2025 The Authors. Published by American Chemical Society 9227 https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 This article is licensed under CC-BY 4.0 Downloaded via UNIV DE SEVILLA on July 4, 2025 at 14:56:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. security crop. Although sorghum is a relatively droughtand heat-tolerant species, it can be affected by several environmental stresses. 5 Therefore, with recurrent adverse agroenvironmental perturbations such as drought becoming more common as the climate continues to change, there is a clear need to improve the sustainability of crop production to ensure the global security of food, feed, and agro-industrial production systems. The development of biotechnologically advanced materials gives rise to new nanofertilizers, nanopesticides, growth enhancers of plants, nanosensors, and nanovectors for active substance transportation. 6 Compared to conventional and chemical priming techniques, nanopriming is a fast-emerging technique offering new research areas to improve plant stress tolerance. 7 Nanoscale material reduction offers benefits such as improved efficiency, durability, reduced nontarget effects, and lower active ingredient use for crop protection, yielding ecological advantages. 8 Among others, metal and metal oxide nanoparticles show both favorable and unfavorable impacts on the physio-biochemical characteristics of plants, depending on concentration and surface functionalization. In the field of nanoagriculture, nanoparticle-mediated seed priming (also known as seed nanopriming) is one of the most explored techniques involving nanotechnology to improve seed germination under hostile conditions. 7 Seed priming is a presowing treatment that consists of treating seeds with suitable nanomaterials before planting to initiate the prerequisite metabolic activities for pregermination without radical protrusion. 7 Indeed, seed is a primary requirement for crop production, which carries the genetic potential of variations and determines the ultimate productivity. However, many studies have demonstrated that high quantities of nanoparticles (e.g., 10−5000 ppm) can have toxic effects on crops, including lettuce, tomato, wheat, and cucumber, being strongly dependent on NPs surface functionalization. 9 Thus, it is necessary to develop nanomaterials with improved properties that boost seed nanopriming, limiting major drawbacks. In this scenario, iron oxide and silver nanoparticles were extensively used to address iron deficiency, boost seed germination, improve drought tolerance, and reduce saltinduced toxicity. 10,11 Besides their biological effects, AgNPs possess peculiar optical properties, the so-called surface plasmon resonance (SPR), whose position in the visible region is strongly sizeand shape-dependent, 12 and they can be easily functionalized with thiols. In the nanoscale size, magnetite and maghemite show peculiar magnetic properties that allow the development of recoverable and reusable systems by applying an external magnetic field. 13 In particular, maghemite is a stable iron oxide with ferromagnetic behavior and superparamagnetic effects in ultrafine particles, 14 with possible functionalization with silanes or amines. In addition to the positive impact of the single components, the preparation of multifunctional hybrid materials allows the incorporation of diverse features in a single nanoplatform. 2,3,15 Fe/Ag nanohybrids such as bimetallic FeAgNPs, 16 Ag@Fe3O4, and Fe2O3@Ag composite nanoparticles, 17,18 and FeOx/ AgNPs core−shell nanoparticles 19 showed superior properties compared with individual components. The copresence of magnetic and electrostatic interactions is a challenging feature, giving rise to aggregation instability. This causes a variation of the NPs’ physicochemical properties, limiting their applicability (especially in the case of naked, uncoated NPs). 17 To prevent their coalescence, common functionalization strategies involve the use of molecular coatings or the use of polymer shells on IONPs, followed by in situ or ex situ chemical reduction of AgNO3. 16,17 They were used primarily in phytotoxicity studies or as antibacterial and antifungal agents. 20 It has been demonstrated that within a certain concentration range (0.1−1000 ppm), AgNPs can act as adjuvants in the early stages of plant development. 21 At a concentration >0.14 mg/kg, AgNPs represent a viable alternative to conventional disinfectants (due to their intrinsic antibacterial and antifungal properties), 22 or they can act as pesticides performing the function of crop disease protection in insect and pathogen management. 7 However, noncovalent interaction between AgNPs and IONPs can limit colloidal stability and material homogeneity with a harmful release into and accumulation in the environment. 23 Thus, improved stability and aqueous dispersibility for Fe/Ag nanosystems (both colloidal and chemical) are needed to take advantage of the synergistic nanohybrid properties. Among others, hydrophilic covalently linked FeOx-AgNPs nanohybrids offer the potential for high colloidal and chemical stability. In this work, water-dispersible maghemite nanoparticles (γFe2O3NPs) were synthesized by a typical Fe3+/Fe2+ coprecipitation method 3 in the presence of (3mercaptopropyl)trimethoxysilane (MPTMS) ligand, containing −SH ending groups to enable the subsequent covalent link with AgNPs. The γ-Fe2O3NPs-MPTMS were isolated and decorated with AgNPs formed in situ by chemical reduction of AgNO3with NaBH4in the presence of sodium 3-mercapto-1propanesulfonate (3MPS) as an additional AgNPs covalent functionalizing agent, improving their colloidal stability and aqueous dispersion. Multifunctional γ-Fe2O3NPs-MPTMSAgNPs-3MPS nanohybrids were obtained by modulating the Ag content during the synthesis procedure, obtaining FeOxAgNPs_1 (in the case of γ-Fe2O3NPs-MPTMS: AgNO3: 3MPS 1:1:10 wt/wt) and FeOx-AgNPs_10 (in the case of γFe2O3NPs-MPTMS: AgNO3: 3MPS 10:1:10 wt/wt). The assynthesized colloidal nanohybrids were tested as seed nanoprimers to evaluate their influence on the Sorghum bicolor (L.) Moench Bianca, (S. bicolor) germination in the 0.1−100 ppm concentration range and compared with naked γ-Fe2O3NPs, pristine γ-Fe2O3NPs-MPTMS, and AgNPs-3MPS. Spectroscopies (UV−Vis, infrared in the midand far-IR regions, Raman), dynamic light scattering (DLS), ζ-potential, powder X-ray diffraction (PXRD), and magnetic susceptibility were used to elucidate the optical, structural, hydrodynamic, and magnetic characteristics of samples. Inductively coupled plasma optical emission spectrometry (ICP-OES) and elemental analysis allowed the evaluation of metal content, while morphological features were investigated by highresolution transmission electron microscopy (HR-TEM). To the best of our knowledge, this is the first report on the synthesis and use of hydrophilic covalent FeOx-AgNPs nanohybrids to promote sorghum seed germination. Although the use of metal-based NPs as a priming agent is not new in agriculture, it is becoming popular in recent times due to the tremendous benefits associated with the use of inorganic sources as nutri-priming agents. Consequently, current research aims to optimize synthesis conditions with extensive morphostructural characterization and evaluate how the different NPs affect Sorghum bicolor (L.) Moench Bianca metabolism through the definition of seed metabolic profile by NMR spectroscopy. According to the results, their effects on sorghum germinated seed development were studied by ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9228 optimizing the Fe/Ag ratio in the nanohybrids, opening promising applications in seed priming practices. 2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. Iron(II) chloride tetrahydrate (FeCl2·4H2O, ≥99.0%, M.W. 198.81 g/mol), iron(III) chloride (FeCl3, 97%, M.W. 162.20 g/mol),silver nitrate (AgNO3,≥99.0%, M.W. 169.87 g/mol), (3-mercaptopropyl)trimethoxysilane (HS- (CH2)3Si(OCH3)3, MPTMS, 95%, M.W. 196.34 g/mol, d (25 °C) = 1.057 g/mL), sodium 3-mercapto-1-propanesulfonate (HS- (CH2)3SO3Na, 3MPS, 90%, M.W. 178.21 g/mol); sodium borohydride (NaBH4,≥98.0%, M.W. 37.83 g/mol), ethanol 96% (CH3CH2OH), and isopropyl alcohol (i-PrOH) were all from Merck (Merk Life Science, Milan, Italy) and used without further purification. Nitric acid Suprapur (HNO3, 65%) and ammonia solution (25%) were from Carlo Erba Reagents (Milan, Italy). Ultrapure water (H2Oup, 18.3 MΩ·cm) was produced with a Zeneer Power I Scholar-UV (Full Tech Instruments, Rome, Italy) water purification system equipped with a 0.01 μm filter and used within 24 h. A commercial nickel (Ni−Cu−Ni)-coated neodymium magnet possessing 88 kg magnetic force, magnetization quality of N52, and 1.42−1.47 T of magnetic strength (magnets4you GmbH, Lohr am Main, Germany) was used to isolate magnetic nanoparticles. 2.2. Synthesis of Naked and MPTMS-Coated γ-Fe2O3 Nanoparticles. Naked γ-Fe2O3NPs were synthesized following a previously published coprecipitation method. 3 Accordingly, in a twoneck round-bottom flask, 0.4000 g of FeCl3(2.47 10−3mol) and 0.2500 g of FeCl2·4H2O (1.26 10−3mol) were dissolved in 25 mL of H2Oup and the mixture degassed with Ar(g) for 15 min. Then, the reaction mixture was heated to 80 °C under reflux, and the pH was adjusted to 11.00 by a fast addition of 10 mL of 10 M NaOH. The solution was vigorously stirred for 1 h and then cooled to room temperature. The dark-brown precipitate containing γ-Fe2O3NPs was magnetically separated and washed with ultrapure water (5×, 160 mL) to remove the excess amount of NaOH and other nonmagnetic species. Yield = (34 ±10) wt % (calculated as dried γ-Fe2O3NPs (g)/ total amount of precursors weight ratio). For the silanization process, 0.1650 g of γ-Fe2O3NPs was dispersed in 30 mL of a 2:1 v/v EtOH: H2Oup mixture and sonicated for 5 min. Subsequently, 15 μL of ammonia solution was added to the colloidal suspension to adjust the pH to 8.00. Then, 25 μL of (3mercaptopropyl)trimethoxysilane (MPTMS, 2.64 10−2g) in a γFe2O3NPs: MPTMS 1:5 weight ratio was added to the mixture. The reaction was allowed to react at room temperature for 24 h. After the reaction, the γ-Fe2O3NPs-MPTMS were magnetically separated and washed with ultrapure water (3×, 60 mL) to remove unbound silane. Purified naked and MPTMS-coated γ-Fe2O3NPs were freeze-dried, stored as solid pellets for further use under an inert Ar atmosphere, and protected from light. Yield = (60 ±14) wt % (calculated as dried γ-Fe2O3NPs-MPTMS/naked γ-Fe2O3NPs weight ratio). Elemental analysis for naked γ-Fe2O3NPs found: C 0.06; H 0.55 ; N 0.00; S 0.00%. Elemental analysis for γ-Fe2O3NPs-MPTMS found: C 2.70; H 0.73 ; N 0.00; S 2.05%. 2.3. Synthesis of 3MPS-Functionalized AgNPs. Hydrophilic AgNPs functionalized with sodium 3-mercapto-1-propanesulfonate (3MPS) were synthesized by optimizing a previously published wet chemical reduction method, and modifications are reported here. 2 0.1000 g of AgNO3(5.89 10−4mol) was dissolved in 10 mL of H2Oup and mixed with 10 mL of 3MPS(aq) (0.4196 g, 2.35 10−3mol) in a AgNO3/3MPS 1:4 molar ratio. The reaction mixture was degassed with Ar for 15 min, and complete nucleation of AgNPs stabilized by 3MPS thiols (hereafter reported as AgNPs-3MPS) was achieved upon the addition of 10 mL of NaBH4(aq) reducing agent (0.2227 g, 5.89 10−3mol, AgNO3:NaBH41:10 mol/mol). The reaction mixture was vigorously stirred for 2 h at room temperature. The as-synthesized AgNPs-3MPS were purified by centrifugation in H2Oup (4 ×30 mL each at 13,400 rpm (17,060g), +8 °C). Then, the precipitate was subjected to dialysis under stirring in ultrapure water for 7 days, replacing the external H2Oup once a day to completely remove unbound thiols and byproducts. Purified AgNPs-3MPS was freezedried and stored as solid pellets for further use under an inert Ar atmosphere and protected from light. Yield (35 ±2) wt % (calculated as AgNPs-3MPS/AgNO3weight ratio). 2.4. Synthesis of Functionalized FeOx-AgNPs Nanohybrids. Hydrophilic FeOx-AgNPs nanohybrids were prepared via a two-step synthesis procedure exploring two different weight ratios, i.e.,γFe2O3NPs-MPTMS: AgNO3: 3MPS 1:1:10 wt/wt (FeOx-AgNPs_1) and γ-Fe2O3NPs-MPTMS: AgNO3: 3MPS 10:1:10 wt/wt (FeOxAgNPs_10). First, 0.0200 g of previously prepared γ-Fe2O3NPsMPTMS was redispersed in 5 mL of H2Oup using an ultrasonic bath for 1 min. Then, 5 mL of AgNO3(0.0050 g for FeOx-AgNPs_1, and 0.0500 g for FeOx-AgNPs_10) and 5 mL of 3MPS (0.4120 g for FeOx-AgNPs_1, and 0.0412 g for FeOx-AgNPs_10) aqueous solution were mixed with γ-Fe2O3NPs-MPTMS suspension and 5 mL of NaBH4(aq) (in a AgNO3/NaBH41:10 molar ratio) as a reducing agent was added dropwise under an Ar atmosphere. After 2 h of reaction, the obtained nanohybrids were magnetically separated and washed repeatedly with ultrapure water (3×, 40 mL) until the complete disappearance of the plasmonic band of unbound AgNPs-3MPS in the supernatant. Samples were freeze-dried under an inert Ar atmosphere and protected from light. Yield = (40 ±9) wt % for FeOx−AgNPs_1 and (71 ±8) wt % for FeOx-AgNPs_10 (calculated as nanohybrid/γFe2O3NPs-MPTMS weight ratio). Elemental analysis for FeOx-AgNPs_1 found: C 2.41%; H 0.63%; N 0.00%; S 3.28% Elemental analysis for FeOx-AgNPs_10 found: C 2.44%; H 0.70%; N 0.00%; S 2.20% 2.5. Preparation and Method of Priming Samples. To investigate the impact of NPs on seed germination, seeds obtained from Sorghum bicolor (L.) Moench genotype Bianca (Padana Sementi s.r.l, Tombolo, Padova) were subjected to surface sterilization for 30 min in a 1.25% sodium hypochlorite solution (150 mL), followed by rinsing with distilled water (H2Od). Subsequently, the seeds were placed in Petri dishes and treated with different NPs concentrations (0.01, 0.1, 1, 10, and 100 ppm). To do so, AgNPs-3MPS, naked γFe2O3NPs, FeOx-AgNPs_1, and FeOx-AgNPs_10 nanohybrids were dispersed in H2Odusing an ultrasonic bath (100 wt, 40 kHz) for 10 min and added to the seeds (3 mL per plate). As a control, the hydropriming was done using H2Od. The plates were incubated at 28 °C for 24 h in the dark, ambient humidity, and then examined. For each concentration, six replicates were considered. Germination was monitored daily until the radicle length surpassed 2 mm as the criterion for germination. The final germination rate percentage (GR %) was determined as the percentage obtained by dividing the number of germinated seeds by the total number of seeds sown. The mean germination time (MGT), the germination speed index (GSI), and the dry weight to fresh weight ratio (DW/FW) were calculated as follows = ×MGT (ni ti)/ (ni) where ti = the number of days since the beginning of the observation and ni = the number of seeds germinated on the i-th day; 24 the germination speed index (GSI) indicates how fast the seeds germinated during an observation period, the reported value is expressed in seeds per day. 25 Univariate analysis was performed by One-way ANOVA test as described below (paragraph 2.7). Germinated seeds were counted and weighed, then dried at 50 °C for 2 days until further analysis. 26 The results of the germination rate were used to select the best samples to study the metabolome by a 1H NMR-based metabolomics approach. 2.6. Characterization Techniques. 2.6.1. UV−Visible Spectroscopy. The UV−vis spectra were recorded in water using a Varian Cary 100 UV−vis spectrophotometer (wavelength range of 200−800 nm) at room temperature. Quartz cells with a 1.5 mL volume and 1 cm optical path length were used. 2.6.2. Dynamic Light Scattering. The hydrodynamic diameters (<2RH>, nm) and ζ-potentials (ζ-pot., mV) of colloidal suspensions ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9229 were measured using a Malvern Zetasizer Nano ZS90 instrument at 25 °C using a 4 mW He−Ne laser light with the wavelength of 632.8 nm and an automatic attenuator. Measurements were done in triplicate (10 runs each) and reported as a mean value ±standard deviation (SD). 2.6.3. Fourier Transform Infrared Spectroscopy. Spectra were acquired on a Bruker Vertex 70 instrument in attenuated total reflectance (ATR) mode in the range of 4000−600 cm−1and in trasmittance mode (4000−400 cm−1)The samples were deposited as a solid powder on the ATR sample holder or deposited from their CHCl3suspensions onto a KRS-5 cell. Far-IR analysis was performed by depositing the samples onto a polyethylene disk (transparency range from 500 to >100 cm−1) from i-PrOH suspensions. Resolution was 4 cm−1with a minimum of 32 scans. 2.6.4. Raman Spectroscopy. Raman spectra were recorded at room temperature, in backscattering geometry, with an inVia Renishaw micro-Raman spectrometer (Wotton-under-Edge, Gloucestershire, UK), using the 514.5 nm emission line from an Ar+ion laser as the exciting source. Repeated accumulations (10 scans ×10 s) were generally acquired on at least four regions of each sample using a 5× objective to check the sample homogeneity. The spectral resolution was 2 cm−1and spectra were calibrated by using the 520.5 cm−1line of a silicon wafer. Spectra processing included baseline removal and curve fitting using a Gauss−Lorentz cross-product function by Peakfit 4.12 (Systat Software Inc., San Jose, CA, USA, 2007). 2.6.5. Nuclear Magnetic Resonance. NMR experiments were carried out on a JNM-ECZ 600R (JEOL Ltd., Tokyo, Japan) spectrometer operating at the proton frequency of 600 MHz and equipped with a multinuclear z-gradient inverse probe head, at 298 K. For the monodimensional 1H experiments of hydrophilic phases, a presaturation pulse sequence for water suppression was used, using a time length of 2 s, a spectral width of 9.03 kHz, and 64k data points, corresponding to an acquisition time of 5.81 s. The pulse length of 90°flip angle was set to 8.3 μs, the recycling delay was set to 5.72 s. Similar parameters were employed for the lipophilic phases, without employing the presaturation sequence for water suppression. Bidimensional 1H−1H TOCSY and 1H−13C HSQC experiments were also carried out for spectral assignment following an already reported procedure. 27 Compound quantities were expressed in mg/ 100g, by comparing with the standard signal and normalized by the number of protons of each signal (for TSP 9 protons, for HMS 18 protons) and further normalized by the fresh weight of the samples. For each sample, approximately 0.5 g was weighed and extracted following an adapted Bligh−Dyer protocol. 26 Prior to extraction, each sample was ground in a mortar under liquid nitrogen and consequently added to 2 mL of cold methanol, followed by 2 mL of cold chloroform and 1.2 mL of water. After each solvent addition, the samples were stirred and stored at 4 °C overnight. Subsequently, they were centrifuged for 30 min at +4 °C, with a rotation speed of 11,000 rpm. Following this step, the upper hydrophilic and lower lipophilic phases were carefully separated and dried under a nitrogen flux. Samples were stored at −80 °C. The hydrophilic phase was resuspended in 0.7 mL of D2O containing 3-(trimethylsilyl)- propionic-2,2,3,3-d4acid sodium salt (TSP, 2 mM), whereas the lipophilic phase was resuspended in 0.7 mL of CDCl3containing hexamethyldisiloxane (HMS, 2 mM), as internal and chemical shift reference standards. 2.6.6. High-Resolution Transmission Electron Microscopy. HRTEM images were acquired using an FEI Talos F200S field emission gun (FEG) microscope operating at 200 keV on samples deposited on a Ni-support grid coated with a carbon amorphous film. Energy dispersive X-ray spectroscopy (EDX) compositional analysis maps were collected by using a Super-X energy dispersive X-ray spectrometry system, which includes two silicon drift detectors coupled to the microscope in the scanning transmission electron microscopy (STEM) mode, using spatial drift correction and a dwell time of 0.2 s. 2.6.7. Inductively Coupled Plasma Optical Emission Spectrometry. Silver (Ag) and iron (Fe) content was quantified using an ICPOES Avio 220 Max instrument (PerkinElmer Inc., USA). For sample digestion, 4 mL of HNO3(65%, VWR Chemicals) was added to each sample in separate 50 mL flasks at 80 °C. After complete dissolution, the volume was adjusted with H2Od. Following acid digestion, samples were diluted 1:10 v/v and 1:100 v/v with H2Odcontaining 2% HNO3v/v. Calibration curves were generated by appropriate dilutions of a multielement standard solution (ICP multielement standard solution IV, Supelco, Merck), with emission intensities recorded at 328.068 nm for Ag and 238.204 nm for Fe, across a concentration range of 0.01−10 ppm. 2.6.8. Powder X-ray Diffraction. Phase analysis was obtained by XRD using a Philips PW 1729 diffractometer equipped with a computer for data acquisition and analysis (software APD-Philips). Scans in the 5−70 2θrange were taken with a 2θstep of 0.01°, using Ni-filtered Cu Kαradiation. 2.6.9. Magnetic Susceptibility Measurements. Magnetic susceptibility measurements at room temperature were carried out on a Sherwood Scientific Ltd. instrument by using as standard HgCo- (SCN)4(χg= 16.44 ×10−6c.g.s. at 20 °C). 28 For the measurements, the Gouy method was used, which allowed us to determine the value of the magnetic susceptibility of a sample by evaluating the difference in weight in the presence and absence of a magnetic field. The sample, packed in a glass tube, was suspended in a nonhomogeneous magnetic field, and the force exerted was determined by the instrument. The force on the sample was proportional to the field gradient as it is not totally immersed in it. For such measurements, it is necessary to use a standard with a known magnetic susceptibility to determine the calibration constant. 2.6.10. Elemental Analysis. Elemental analyses (C, H, N, S) were done using the EA 1110 Carlo Erba instrument available at the Chemistry Department of Sapienza University of Rome. 2.7. Statistical Analysis. MATLAB R2023a (MathWorks, Natick, Massachusetts, USA) with the Statistics and Machine Learning Toolbox package was used as the software for the univariate and multivariate analysis, with a home-built script. The multivariate analysis was carried out employing an ANOVA-simultaneous component analysis (ASCA) function. 29 Prior to analysis, data were mean-centered and autoscaled in order to equalize the importance of the variation of each variable. ASCA combines multivariate data with the design experiment of ANOVA and operates by partitioning the variance of the main matrix in different effect matrices, as reported in the following equation = +X X E A Herein, the original matrix Xis decomposed into factors considered by the design experiment. For this analysis, only the NPs concentration was codified as the factor XA, as for the matrix Ethe rest of the variance was included. For the SCA part, the number of Simultaneous Components utilized by the model is due to the number of different groups of samples considered by the experimental design. In this work, since one control group and 5 different treatments were used, the factor’s maximum rank was 5. A bootstrapping procedure was also employed for the calculation of confidence intervals of the SC loadings, estimated by 10000 bootstrap repetitions. 30 The ASCA model significance for factor XAwas estimated by a permutation test of the contribution of the factor by the sum-of-squares (SSQ). The significance was estimated by 10,000 randomizations and by controlling if the SSQ of the experimental data were outside the 95th percentile of the distribution. 31 For the univariate analysis, the normality and homoscedasticity of each variable distribution were evaluated using the Shapiro−Wilk test and Brown Forsythe, respectively. 32 If those conditions were respected, One-way ANOVA was employed; otherwise, the nonparametric Kruskal−Wallis test was used. 33 The multiple comparison test was corrected with Bonferroni, 34 considering a significance of p< 0.05. 3. RESULTS AND DISCUSSION 3.1. Synthesis of Functionalized Iron Oxide-Silver Nanohybrids. 3.1.1. Spectroscopic Characterization. Iron oxide-based nanomaterials, i.e., naked γ-Fe2O3NPs, γACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9230 Fe2O3NPs-MPTMS, and γ-Fe2O3NPs-MPTMS-AgNPs-3MPS nanohybrids, i.e., FeOx-AgNPs_1 and FeOx-AgNPs_10, were synthesized via a combination of coprecipitation method (used for naked γ-Fe2O3NPs and γ-Fe2O3NPs-MPTMS) followed by an in situ wet chemical reduction of Ag+metal precursor in the presence of 3MPS as an additional hydrophilic functionalizing agent (used for nanohybrid synthesis), as illustrated in Figure 1a. The two different weight ratios (γ-Fe2O3NPs-MPTMS:AgNO3:3MPS = 1:1:10 for FeOx-AgNPs_1 and 10:1:10 for FeOx-AgNPs_10) were selected to modulate the silver content on the nanohybrid surface, according to a previous study. 2 This strategy allowed for the control of the degree of silver content in the final nanohybrid. For comparison, AgNPs-3MPS were synthesized according to the literature. 2 In the UV−vis spectra (Figure 1b), all the synthesized samples showed the absence of FeCl2·4H2O and FeCl3(precursors) absorption bands (Figure S1) and the presence of a broad absorption centered at 355 nm associated with O(2p) →Fe(3d) electronic transitions indicating the formation of γ-Fe2O3NPs. 3 In the case of nanohybrids, a characteristic plasmonic band was observed at about 480 nm, resulting from the contribution of surface plasmon resonance (SPR) phenomenon typical of AgNPs, as evidenced by the SPR of pristine AgNPs-3MPS. Compared with isolated AgNPs-3MPS, in the nanohybrids the SPR band resulted in a 38 nm redshift (SPRAgNPs = 442 nm, SPRnanohybrids = 480 nm) due to the presence of high refractive index γFe2O3NPs, reflecting the proximity of plasmonic nanoparticles with the metal oxide surface. 35 The plasmonic band was more pronounced in the case of FeOx-AgNPs_1, attributed to the higher concentration of AgNO3precursor used during the synthesis procedure, which in turn led to a higher AgNPs yield. 3.1.2. Elemental Composition and Magnetic Properties. The metal quantification in the nanohybrids was carried out with ICP-OES and demonstrated a ca. 30% increase in the Ag content for FeOx-AgNPs_1 (Ag = 84.3 ±0.8 ppm) compared with FeOx-AgNPs_10 (Ag = 22.7 ±0.2 ppm), corresponding to (15 ±1) wt % and (4 ±1) wt %, respectively (Figure 1c). The iron (Fe) was found to be (245 ±1) ppm for FeOxAgNPs_1 and (339 ±7) ppm FeOx-AgNPs_10 (i.e., (45 ±1) and (56 ±2) wt %, respectively). Therefore, the quantity of nanostructured Ag on the maghemite surface (which increased with increasing the AgNO3precursor concentration) can be tuned by modifying the stoichiometry during the synthesis phase, as reported for similar Ag-decorated nanosystems. 2 The oxygen (O) content from ICP-OES was obtained by difference considering a negligible contribution from carbon (C), sulfur (S), and hydrogen (H) elements (as obtained from the elemental analysis, see paragraph 2.4), being 219 ±2 ppm (40 ±1 wt %) for FeOx-AgNPs_1 and 241 ±2 ppm (40 ±1 wt %) for FeOx-AgNPs_10, showing a slight variation between the two nanohybrids. Figure 1. (a) Schematic representation of synthesis procedure adopted for: γ-Fe2O3NPs-MPTMS, and FeOx-AgNPs nanohybrids. (b) UV−visible spectra in H2Oup of γ-Fe2O3NPs (black line), silanized γ-Fe2O3NPs (dark cyan line), FeOx-AgNPs_1 (red line), and FeOx-AgNPs_10 (blue line). (c) ICP-OES measurements expressed as concentration (ppm) and %wt (on column) of Ag, Fe, O in the FeOx-AgNPs nanohybrids. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9231 Qualitative magnetic characterization was carried out, and data are listed in Table 1. The high magnetic susceptibility values per gram measured for the four compounds were indicative of ferromagnetic behavior. Due to the higher diamagnetic (Ag) content in the FeOx-AgNPs_1 sample, the measured χgvalue was lower than that of the FeOx-AgNPs_10 sample, where Ag was about 4 times lower, as experimentally measured by ICP-OES (Figure 1c). These results suggest that all compounds have ferromagnetic behavior. The increase in the diamagnetic contribution caused a decrease in the magnetic susceptibility value. 3.1.3. Colloidal Stability and Hydrodynamic Properties. Hydrodynamic size (<2RH>, nm) and ζ-potential (mV) were evaluated by DLS on colloidal aqueous suspensions, and the results obtained are summarized in Table 1 and Figure S2. Nanohybrids showed similar diameters with quite uniform size distribution (PDI ca. 0.2). Despite the different amounts of AgNPs-3MPS linked on the γ-Fe2O3NPs surface, in colloidal suspension, the diameter seems to be mainly governed by iron oxide nanoparticles size. Indeed, compared with naked monometallic γ-Fe2O3NPs, a lower <2RH> was found. Naked γ-Fe2O3NPs displayed a single population with a diameter of (280 ±135) nm and an incipient instability with ζ-potential = (−13 ±4) mV, higher than γ-Fe2O3NPs-MPTMS with <2RH> = (243 ±135) nm. In the latter, the decrease in the particle size and improved colloidal stability (ζ-potential = (−26 ±4) mV) was due to the presence of the MPTMS stabilizing layer around the metal core, which is reported to tune the aggregation and sedimentation behavior of colloidal metal oxide nanoparticles. 36 More importantly, the −SH ending groups of MPTMS allowed a functionalization with additional hydrophilic AgNPs-3MPS nanostructures, further reducing the size up to <2RH> = (235 ±120) nm for both nanohybrids. The presence of the sulfonate-ending moiety (−SO3 −) of 3MPS on AgNPs chemically linked to γ-Fe2O3NPs-MPTMS resulted in a negative value of ζ-potential ≥ − 22 mV, highlighting the colloidal stability of nanohybrids via a combination of both steric and electrostatic interactions provided by the MPTMS and 3MPS functionalizing agents. 3.2. Structural Characterization. 3.2.1. Powder X-ray Diffraction. Phase analysis of the synthesized nanoparticles and hybrids was based on XRD patterns, as reported in Figure Table 1. Naked γ-Fe2O3NPs, γ-Fe2O3NPs-MPTMS, FeOx-AgNPs_1, and FeOx-AgNPs_10 characterizations: hydrodynamic diameter (<2RH>, nm), polydispersity index (PDI) and ζ-potential (mV) values obtained by DLS analysis on colloidal dispersions in H2Oup, and magnetic susceptibility (χg, cgs) values on powders Sample name <2RH> (nm) PDI ζ-pot (mV) χg(cgs) FeOx-AgNPs_1 235 ±120 0.225 ±0.003 −22 ±4 2.732·10−4 FeOx-AgNPs_10 236 ±120 0.262 ±0.001 −26 ±6 4.281·10−4 γ-Fe2O3NPs-MPTMS 243 ±135 0.28 ±0.02 −26 ±4 4.093·10−4 γ-Fe2O3NPs 280 ±135 0.36 ±0.03 −13 ±4 4.288·10−4 Figure 2. (a) Powder XRD patterns obtained for γ-Fe2O3NPs-MPTMS (dark cyan), FeOx-AgNPs_10 (blue line), FeOx-AgNPs_1 (red line), and AgNPs-3MPS (magenta line). Maghemite and metallic Ag are indexed. (b) Mid-Infrared spectra (4000−400 cm−1) for FeOx-AgNPs_1 and FeOxAgNPs_10. (c) Far-IR spectra (600−400 cm−1) for FeOx-AgNPs_1 and FeOx-AgNPs_10. (d) Raman spectra of FeOx-AgNPs_1 and FeOxAgNPs_10. ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9232 2a. All iron oxide-based nanomaterials showed the presence of maghemite (γ-Fe2O3, card number 25−1402, JCPDS International Centre for Diffraction Data, 1601 Park Lane, Swarthmore, Pennsylvania 19801, USA) with reflections of the tetragonal system at 2θ: 30.3 (hkl 206), 35.7 (hkl 119), 43.3 (hkl 0012) 57.4 (hkl 1115), 63.0 (hkl 4012). XRD pattern of naked γ-Fe2O3NPs is reported in Figure S3. AgNPs-3MPS nanoparticles revealed the presence of metallic Ag (card number 4−783, JCPDS International Centre for Diffraction Data, 1601 Park Lane, Swarthmore, Pennsylvania 19801, USA) with reflections of the cubic system at 2θ: 38.1 (hkl 111), 44.2 (hkl 200), 64.5 (hkl 220). As to the functionalized nanohybrids, both FeOx-AgNPs_1 and FeOxAgNPs_10 samples showed X-ray reflections of maghemite, while only FeOx-AgNPs_1 revealed the presence of metallic silver. 3.2.2. Mid-infrared Spectroscopy. Chemical characterization of surface functional groups of γ-Fe2O3NPs-MPTMS and related nanohybrids was carried out via FTIR and far-IR analysis on dried samples. Mid-IR spectra of naked γFe2O3NPs, γ-Fe2O3NPs-MPTMS, and AgNPs-3MPS are reported in Figures S4−S6. Figure 3. Morpho-structural observations of γ-Fe2O3NPs-MPTMS interacting with AgNPs-3MPS nanohybrid. (a) BF-TEM image of γ-Fe2O3NPsMPTMS. Inset: EDX (Fe, Si, O, S). a-I IFFT high-resolution image of region I (blue square). a-II high-resolution IFFT image of region II (blue square). (b) EDP taken from (a). (c) BF-TEM image of FeOx-AgNPs_1. Inset: the EDX (Ag, Fe, S, Si, O).c-I FFT pattern of region I (green square) within hexagonal shape of γ-Fe2O3NPs and singles spots of AgNPs-3MPS (silver circles); the corresponding high-resolution IFFT image illustrating a superposition of nanocrystal lattice fringes and IFFT image of AgNPs-3MPS by subtracting γ-Fe2O3NPs contribution (below). c-II FFT pattern of region II (green square) with superposition of γ-Fe2O3NPs hexagonal and cubic shapes and singles spots of AgNPs-3MPS (silver circles), the corresponding high-resolution IFFT images with AgNPs-3MPS nanocrystal lattice fringes without γ-Fe2O3NPs contribution (below). ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9233 The FTIR spectra of FeOx-AgNPs_1 and FeOx-AgNPs_10 are shown in Figure 2b. Both samples showed the specific Fe− O tensile bands of naked maghemite (Figure S4) at 450 and 582 cm−1(T1 modes) and 632 cm−1related to its spinel structure, of which the band at lower wavenumber (∼400 cm−1) was attributed to the inherent vibration of octahedral groups, whereas the one at ∼600 cm−1was associated with tetrahedral locations. 3,37 The band centered at 582 cm−1 (symmetrical Fe−O stretching) splits into two characteristic vibrations due to the creation of vacancy defects and the vanishing of the Fe(II) ion from the octahedral sites upon the formation of γ-Fe2O3phase. 3 The fingerprint region (1300− 750 cm−1) was the most representative of nanohybrid formation, reflecting their stoichiometry. Indeed, in the case of FeOx-AgNPs_10, the spectrum was dominated by MPTMS signals, showing characteristic stretching (ν) absorption bands of Si−O−Si (1000 cm−1), Si−OH (1112 cm−1), and CH2 wagging (1244 cm−1) 38 vibration, with similar intensities to those of γ-Fe2O3NPs-MPTMS (Figure S5). The presence of silanol (Si−OH) groups accounts for different silane binding modes on the metallic surface, thus ensuring its hydrophilicity. Notably, the weak signal at 1043 cm−1was attributed to the symmetric stretching (νs) vibration of the sulfonate (−SO3 −) ending moiety, as found in pristine AgNPs-3MPS (Figure S6). 2 Conversely, at higher AgNPs-3MPS decoration (FeOxAgNPs_1 nanohybrid), signals related to 3MPS functionalizing thiol predominate over the MPTMS one. FeOx-AgNPs_1 nanohybrid spectrum showed only two signals in this spectral region, both related to stretching modes of the −SO3 −end group: the symmetric νs= 1045 cm−1vibration and its asymmetric counterpart νas = 1175 cm−1. Vibration bands related to MPTMS appeared as a shoulder of these bands (at ∼1117 and ∼1000 cm−1), contributing to their broadening. The 3000−2800 cm−1wavenumber region further corroborates the presence of aliphatic chains with bands at 2955 cm−1 (νas(−CH3)), 2916 cm−1(νas(−CH2)) and 2852 cm−1 (νs(−CH2)). 3.2.3. Far-Infrared Spectroscopy (Far-IR). A better insight into covalent bonds occurring in the nanohybrid structure is spotted in the far-infrared region (600−400 cm−1) reported in Figure 2c. The broad vibration centered at 375 cm−1was due to an additional Fe−O stretching T1mode, 3 which is more pronounced in naked γ-Fe2O3NPs (Figure S4). Indeed, maghemite vibrations in this spectral region combine with those of the free MPTMS functionalizing ligand (Figures S5− S6) and pristine AgNPs-3MPS (Figure S6). Additional IRactive vibrations associated with maghemite structure arose at 212 and 275 cm−1, although of little analytical relevance. 39 Metal−sulfur ν(Ag−S) bond stretching frequencies with a covalent character lie in the range 295−260 cm−1 40 , which in the nanohybrids corresponds to multiple signals centered at ca. 280 cm−1, based on the comparison with the intense signal observed at ca. 278 cm−1in pristine AgNPs-3MPS (Figure S6). It is worth noting that in FeOx-AgNPs_1 and FeOxAgNPs_10, two different Ag−S covalent bonds exist related to the bond with the terminal −SH of MPTMS on one side and the terminal −SH of 3MPS on the other side, which cannot be accurately discriminated. 3.2.4. Raman Spectroscopy. Raman spectra on FeOxAgNPs_1 and FeOx-AgNPs_10 after background subtraction and curve fitting procedure are shown in Figure 2d (range of interest 200−2000 cm−1). Experimental spectra can be found in Figure S7. In both spectra, components in the 340−700 cm−1wavenumber region, 340 (T1mode), 511 (E mode), 676 cm−1(A1mode) for FeOx-AgNPs_1; 364 (T1mode), 487 (E mode), 683 cm−1(A1mode) for FeOx-AgNPs_10) confirmed the formation of pure maghemite γ-Fe2O3phase during synthesis, as already reported for a similar system. 3 In the FeOx-AgNPs_1 spectrum, the typical stretching vibration ν(CC) was found at 1003 cm−1. Aliphatic (−CH2) wagging (ω) appeared at 1366 cm−1and 1220 cm−1(ω), whereas bending vibrations of δ(−CH3) at 1439 cm−1were found. 41 An additional signal at 1539 cm−1was assigned to identify the maghemite phase. 42 The FeOx-AgNPs_10 showed similar absorption at 981 cm−1ν(CC), 1414 cm−1δ(CH2), and 1246 cm−1ω(CH2), although an additional band was found at 1097 cm−1attributable to νa(Si−O−C) partially overlapping with the νs(−SO3 −) of 3MPS 41,43 . The 1320 cm−1mode was assigned to Raman-active phonons of the Fe2O3phase. 44 Interestingly, in the FeOx-AgNPs_10, the signal observed at 281 cm−1can be assigned to Ag−S vibration, 43 which is in agreement with the far-IR results (see paragraph 3.2.3). 3.3. Morphological Characterization. The self-assembly behavior of γ-Fe2O3NPs-MPTMS interacting with AgNPs3MPS was investigated using a combination of electron microscopy (HR-TEM, SAED, FFT, IFFT and EDX) and image processing techniques (Figure 3). The morphostructural study of the γ-Fe2O3NPs-MPTMS showed nanoparticles of about 7 nm in size identified in their chemical composition (Figure 3a). The EDX spectroscopy evidenced the presence of intense peaks due to iron oxide with the further appearance of silicon and sulfur species due to the MPTMS ligand (Inset). To ascertain the presence of different phases by enhancing the contribution deriving from small nanocrystals embedded in the structure, the FFT analysis was performed on selected nanoareas of the image. By scanning FFT of Figure 3a, the high-resolution IFFT image of the representative region I, evidenced the crystalline nature of five iron oxide nanoparticles with the lattice spacing d313 = 0.253 nm, belonging to the P43212 tetragonal symmetry of maghemite phase (γ-Fe2O3). Similarly, nanoparticles with the same lattice spacing are shown in region II, and one of them shows a single nanoparticle oriented along the (206) crystallographic plane with a spacing of 0.296 nm. Further confirmation of the γ-Fe2O3formation was evidenced by the electron diffraction pattern of Figure 3a. By measuring the d-spacing of the diffraction rings produced by random orientation, it was possible to identify the γFe2O3NPs of a tetragonal symmetry with preferential crystallographic orientation (313) and its higher-order diffraction ring (516) (Figure 3b). The morpho-structural investigation of the FeOx-AgNPs_1 sample resulted in a nanohybrid arrangement into a superaggregate network (Figure 3c). 45 The presence of the expected chemical species was confirmed by EDX spectroscopy (Ag, Fe, S, Si, and O, bottom-left-side). By FFT scanning imaging, a hexagonal pattern was revealed in the selected image of representative nano region I (green square), belonging to the γ-Fe2O3phase-oriented along the [221] zone axis (left-side). An extra array of diffraction spots with low intensities was present (silver circles). High-resolution IFFT image evidenced crystalline lattice fringes with different orientations with the preferential presence of the lattice spacing d313 = 0.253 nm of the maghemite phase. To highlight the low scattering signal of the silver nanostructure, the contribution of the γ-Fe2O3crystal to the FFT was subtracted before performing the inverse FFT (IFFT). 46 By this procedure, evidence of the presence of small ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9234 silver nanoparticles surrounding the γ-Fe2O3NP is shown below (highlighted by white dotted circles), having a structure with a spacing of 0.235 nm, oriented along the (111) crystallographic plane. The representative nano region II exhibits an FFT pattern with a superposing of the hexagonal (white line, [211]) and cubic (white circles, [211]) shapes of the γ-Fe2O3nanocrystals and diffraction spots of low intensity were still present (silver circles, right-side of Figure 3c). The corresponding IFFT image shows nanocrystals with main lattice spacing d313 = 0.253 nm and the silver lattice fringes d111 = 0.235 nm in the IFFT image below that still surround the γFe2O3nanocrystals. 3.4. Germination Rates and Metabolomic Analyses. 3.4.1. Germination Rates of Sorghum bicolor (L.) Moench Bianca Seeds. The as-synthesized nanocolloids were tested as nanoprimers in Sorghum bicolor (L.) Moench Bianca seeds germination framework. Results of germination rates (GR %) after treatment of sorghum seeds with NPs in the 0.01−100 ppm concentration range are reported in Table 2. Compared to the controls, treatment with hydrophilic AgNPs-3MPS increased the GR % only at 0.01 and 10 ppm. In the case of naked γ-Fe2O3NPs, all concentrations, except 1 ppm, showed a positive impact on the GR %. FeOx-AgNPs nanohybrids showed a similar trend, although some differences could be evidenced. Specifically, major differences were found at low concentrations: 0.01, 0.1, and 1 ppm, compared to control groups and pristine counterparts, in which the highest germination rates (92.5−95.0%) were obtained. Such behavior can be ascribed to a different nanohybrid penetration of the seed coat. Indeed, an increase in water absorption by seeds was reported to have a positive influence on germination mechanisms 47 . Despite their known antibacterial activity, AgNPs can also exert antioxidant properties (promotion of seed antioxidant systems, reduction of ROS content at a certain concentration threshold). 47 Thus, based on the results obtained, it is reasonable to assume that the FeOx-AgNPs_1 nanohybrid (containing the higher amount of AgNPs-3MPS) at the lower concentrations improves the seed abilities to absorb and utilize water while reducing oxidative stress, according to the literature. 48 A comparison with the already published work on Sorghum bicolor germination is reported in Table 3. Since the FeOx-AgNPs_1 nanohybrid showed the best results on the germination rate at the lowest concentrations (0.01−1 ppm), this system was selected to better investigate its role in seed germination and metabolomics analysis. 3.4.2. Nanopriming Effect of FeOx-AgNPs_1 Nanohybrid. To examine the effect of nanopriming with FeOx-AgNPs_1, mean germination time (MGT), seed germination rate (GR %), germination seed index (GSI), and dried weight/fresh weight (DW/FW) were assessed, and results are reported in Figure 4 (full data in Table S1). No significant differences in the mean germination time (MGT) were observed. Significant differences regarding the germination rates (GR %) were observed. In particular, seeds treated with FeOx-AgNPs_1 at 0.01, 0.1, 1, and 100 ppm showed a higher GR % compared to FeOx-AgNPs_1 at 10 ppm, suggesting that the latter concentration is less effective at enhancing rapid seed germination. In addition, seeds treated with FeOx-AgNPs_1 at 10 ppm showed the lowest GSI (23 ±2) compared with unprimed seeds. A significantly lower DW/FW was observed in seeds treated with FeOx-AgNPs_1 at 10 ppm but also at 100 ppm compared to that in unprimed seeds, suggesting that higher nanoparticle concentrations facilitate the permeation of water through the seed coat. Some reports indicate that nanoparticles use the intercellular spaces of the tissues or create new pores mainly through the upregulation of aquaporin production to promote robust seedling germination and growth. 52 Although there was no correlation between the increase in seed water content and the enhancement of germination rate at higher FeOx-AgNPs_1 concentration, a potential time-dependent effect of nanopriming cannot be excluded and could be visible at longer phases of seedling development. The molecular mechanisms underlying seed changes during and after nanopriming treatment remain elusive. 53 As proposed, priming, by constraining water movement, may extend the lag phase and postpone the onset of the log phase of seed germination. 54 The morphological characteristics of the sorghum nanoprimed seeds were normal without signs of induced toxicity (Figure S8). 3.5. Metabolomics Analysis of S. bicolor Seeds Treated with FeOx-AgNPs_1 Nanohybrid. 3.5.1. 1H NMR Metabolome of S. bicolor Seeds Treated with FeOx-AgNPs_1 Nanohybrid. 1H NMR spectra of hydrophilic and lipophilic fractions of seeds treated with the FeOx-AgNPs_1 nanohybrid are reported in Figures S9−S16. A total of 45 metabolites were identified and quantified. Resonance assignment was carried out employing literature and database data 55,56 together with TOCSY and HSQC experiments (Figures S17−S19). ChemTable 2. Percentage of Germination Rate (GR %) Obtained for Different Samples in the 0.01−100 ppm Concentration Range a germination rate (GR %) NPs type 0.01 ppm 0.1 ppm 1 ppm 10 ppm 100 ppm AgNPs3MPS 88 ±18 72 ±18 80 ±7 95 ±7 78 ±4 Naked γ-Fe2O3NPs 88 ±4 92 ±4 72 ±18 95 ±0 88 ±11 FeOx-AgNPs_1 92 ±4 95 ±7 95 ±0 80 ±7 85 ±7 FeOx-AgNPs_10 92 ±11 80 ±14 82 ±4 85 ±14 85 ±0 control (unprimed treatment): 88.5 ±0.2 a Data is presented as mean percentage (±SD) of six biological replicates containing 30 seeds for each S. bicolor treatment. Table 3. Comparison Table with Recent Nanomaterials Used in Seed Priming of S. bicolor Seeds nanomaterial effective concentration (ppm) germination rate (GR %) refs commercial naked nanoiron oxide (n-Fe2O3)10 >90% 49 commercial graphene nanoplatelets 50 19.78% 50 commercial multiwalled carbon nanotubes (CNTs) 50 21.89% 50 naked zinc oxide nanoparticles (ZnONPs) 1000 96.42% 51 FeOx-AgNPs_1 nanohybrid 0.01−1 92−95% this work ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9235 Peculiarities of Hybrid Au/Nanodiamond Engineered Nanostructures. Sci. Rep. 2016,6, 31163. (47) Almutairi, Z. M.; Alharbi, A. Effect of Silver Nanoparticles on Seed Germination of Crop Plants. Nucl. Quant. Eng. 2015,9, 689− 693. (48) El-Saadony, M. T.; Saad, A. M.; Soliman, S. M.; Salem, H. M.; Desoky, E.-S. M.; Babalghith, A. O.; El-Tahan, A. M.; Ibrahim, O. M.; Ebrahim, A. A. M.; Abd El-Mageed, T. A.; Elrys, A. S.; Elbadawi, A. 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ACS Applied Nano Materials www.acsanm.org Article https://doi.org/10.1021/acsanm.5c00702 ACS Appl. Nano Mater. 2025, 8, 9227−9242 9242