Antimicrobial activity of amino-modified cellulose nanofibrils decorated with silver nanoparticles

Antimicrobial activity of amino-modified cellulose nanofibrils decorated with silver nanoparticles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimicrobial activity of amino-modified cellulose nanofibrils decorated with silver nanoparticles Vesna Lazic, Jovan M. Nedeljković, Vanja Kokol This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4507463/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The silver nanoparticles (Ag NPs) conjugated with amino-functionalized cellulose nanofibrils (NH 2 − CNFs) were in situ prepared by reducing silver ions with free amino groups from NH 2 − CNFs. The spectroscopy and transmission electron microscopy measurements confirmed the presence of non-agglomerated nanometer-in-size Ag NPs within micrometer-large NH 2 − CNFs of high (20 wt.-%) content. Although the consumption of amino groups during the formation of Ag NPs lowers the ζ-potential and surface charge of prepared inorganic-organic hybrids (from + 31.3 to + 19.9 mV and from 2.4 to 1.0 mmol/g at pH 7, respectively), their values are sufficiently positive to ensure electrostatic interaction with negatively charged cell walls of pathogens in acidic and slightly (up to pH ~ 8.5) alkaline solutions. The antimicrobial activity of hybrid microparticles against various pathogens ( Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , and Candida albicans ) is comparable with pristine NH 2 − CNFs. However, a long-timescale use of hybrids ensures the slow and controlled release of Ag + ions to surrounding media (less than 1 wt.-% for one month). Amino-modified cellulose nanofibrils Silver nanoparticles Hybrid microparticles Zeta-potential Antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Nanocellulose (NC) is one of the most promising eco-friendly, non-toxic materials, obtained from renewable sources (plants, algae, tunicates, and some bacteria), with the potential to replace synthetic polymers in many applications due to their unique properties, including easy processability and the possibility to tailor morphology, specific surface area, crystallinity, and biocompatibility [ 1 ]. Because of these unique features, NC is recognized as a suitable material for various current and emerging applications, such as nanocomposites, adhesives, paints, barrier and functional coatings, fibers and filaments, films and foils, membranes and filters, biomedical and pharmaceutical products, supercapacitors, and batteries [ 1 – 4 ]. However, pristine NC does not display antimicrobial activity, and with the knowledge of the importance of non-toxic biodegradation for the environment, various approaches were proposed to obtain its antimicrobial activity. Generally, two different approach types are recognizable, the first based on surface modification of NC with aldehyde, amino, and quaternary ammonium groups [ 5 ], and the second based on conjugation with either metal (Au, Ag, and Cu) [ 6 – 8 ], metal oxide nanoparticles (CuO, MgO, ZnO, and TiO 2 ) [ 9 – 13 ], biocidal agents, such as gentamicin [ 14 – 16 ], or antimicrobial polymers [ 17 – 19 ]. Functionalization of NC with amino groups is of particular interest. On one side, amino-functionalized cellulose nanofibrils (NH 2 − CNFs) display antimicrobial activity due to the electrostatic interaction between positively charged amino groups and negatively charged cell walls that enhanced their contact with microorganisms [ 20 – 22 ]. On the other side, the free amino groups are capable of reducing Ag + ions to their metallic form, providing the possibility to prepare free-standing colloidal Ag NPs [ 23 , 24 ] or Ag NPs linked to different types of supports, polymer [ 25 – 27 ] and inorganic [ 28 , 29 ], with high antimicrobial activity. Besides the antimicrobial activity of Ag NPs on support, when they are easily accessible to microbial species in the surrounding medium, Ag NPs dispersed within a polymer matrix also have antimicrobial activity. Recently, we demonstrated that thin films prepared from cellulose nanofibrils loaded with less than 0.5 wt.-% of a nanometer in size Ag NPs inhibited the growth of E. coli after five repeated cycles, indicating that might serve as a sustainable replacement for their synthetic counterparts in food packaging applications [ 30 ]. This study thus aims to evaluate the antimicrobial activity of hybrid based on amino-functionalized cellulose nanofibrils (NH 2 − CNFs) decorated with Ag NPs, prepared in situ , taking advantage of the reducing ability of free amino groups. So, combining inorganic (Ag NPs) and organic (NH 2 − CNFs) components into a hybrid (Ag/NH 2 − CNFs), with the knowledge that each of the components displays antimicrobial activity [ 30 – 32 ], was intended to improve the antimicrobial performance of hybrid under long time working conditions. Samples were characterized in detail before determination of their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against different microbial species (Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa , Gram-positive bacterium Staphylococcus aureus , and fungus Candida albicans ). In addition, the content of the inorganic phase was determined by thermogravimetric measurements, an optical property of hybrid originating from Ag NPs was correlated with transmission electron microscopy data, and X-ray diffraction analysis provided the influence of the inorganic component on the crystallinity of NH 2 − CNFs. Finally, as a crucial factor influencing the electrostatic interaction of samples with bacteria’s wall, pH-dependent total charge and ζ-potentials of samples in different media were determined, including the hydrodynamic size of the hybrids. 2. Experimental 2.1. Materials The wood-based chain-like cellulose nanofibrils (CNFs) with diameters in the 10–70 nm range and length of a few micrometers (1–3 µm) were prepared from bleached softwood pulp, refined in a multistage disc refiner by the University of Maine, The Process Development Center in the USA. Hexamethylenediamine (HMDA, 98% purity) and all other chemicals used were purchased from Sigma-Aldrich Co. Ltd. (USA) and used without further purification. 2.2. Preparation of amino-functionalized CNFs (NH 2 − CNFs) decorated with silver nanoparticles (Ag NPs) First, the amino-functionalized CNFs (NH 2 − CNFs) were prepared by a two-step reaction, as presented in Scheme 1 [ 32 ]. The aqueous dispersion of CNFs (1 wt.-%, 100 mL) was first oxidized to exhibit aldehyde functional groups at the C2 and C3 cellulose units positions by mixing it with pre-dissolved sodium periodate (1.6 g NaIO 4 per 1 g of CNF) and stirred for 48 h in the dark at room temperature. The product was washed thoroughly a few times with deionized water for up to 3 days. The functionalization of pre-oxidized CNFs with HMDA was performed through a Schiff-base reaction via aldehyde groups. For that purpose, 200 ml of 0.5 wt.-% pre-oxidized CNFs suspension was ultrasonicated for 5 min, and then 8 mmol of HMDA was added. The mixture was stirred continuously for 6 h at 30°C, followed by the in-situ reduction of the resulting imine intermediate at room temperature, employing 0.58 g of NaBH 4 . After stirring for 3 h, the products were washed thoroughly several times with deionized water until a neutral pH was reached and used as prepared. In the next step, the Ag NPs were grown onto NH 2 − CNFs by the in-situ reduction of Ag + ions, taking advantage of the reducing ability of free amino groups, which are present in the amino-functionalized CNFs [ 27 ]. Briefly, the mixture having a 1:1 molar ratio between Ag + ions and amino groups (85 mg of AgNO 3 and 2.1 mL of amino-functionalized CNFs in 50 mL of H 2 O) was stirred overnight under reflux at 60°C. The appearance of a yellow-brown color indicated the successful reduction of Ag + ions to the metallic silver. Then, the dispersion was washed several times with deionized water and dried at 40°C in a vacuum oven for 24 h. For clarity reasons, Ag NPs conjugated with NH 2 − CNFs will be denoted further in the text as Ag/NH 2 − CNFs. 2.3. Characterization of NH 2 − CNFs and Ag/NH 2 − CNFs Absorption of NH 2 − CNFs and Ag/NH 2 − CNFs were evaluated in UV–Vis–NIR spectral range by diffuse reflectance measurements (Shimadzu UV–Visible UV-2600 spectrophotometer equipped with an integrated sphere ISR-2600 Plus). The content of metallic silver in Ag/NH 2 − CNFs was determined by thermogravimetric analysis (TGA) using a Setaram Setsys Evolution-1750 instrument. The TGA measurements were performed under a dynamic air atmosphere (flow rate of 20 cm 3 /min) in the temperature range from room temperature to 1000°C (heating rate of 10°C/min). The content of released silver (Ag + ions and Ag NPs) after 30 days of Ag/NH 2 − CNFs incubation in distilled water at room temperature was determined using inductively coupled plasma optic emission spectroscopy (ICP-OES Thermo Scientific iCAP 7400). X-ray diffraction (XRD) measurements of prepared samples were carried out using a Rigaku SmartLab instrument with Cu Kα1,2 radiation. The measurements were performed with continuous scanning at 2 °/min and by collecting the data at 0.02° intervals. Microstructural characterization of the Ag/NH 2 − CNFs was performed on a transmission electron microscope (TEM) JEOL JEM-2100 LaB6 operating at 200 kV. TEM images were acquired with a Gatan Orius CCD camera at 2× binning. Potentiometric titrations of native and modified CNF samples suspended in miliQ water were performed to quantify the process-dependent surface charge contributions. The titration was carried out using a dual-burette instrument (Mettler Toledo T-70) equipped with a combined glass electrode (Mettler TDG 117) and filled with 0.1 M HCl (Merck, Titrisol) and 0.1 M KOH (Baker, Dilut-it). Samples soaked in milli-Q water were rinsed in low pH (0.01 M HCl) to convert the basic and acidic groups into protonated forms and then dried at 40°C. The titrations were carried out at room temperature (23 ± 1°C), forward and backward between pH 2 and 11. The molar concentration, related to the overall charge of the side groups, was calculated from the potentiometric titration data. All the reported values are the mean values of duplicate determinations. The hydrodynamic size and zeta-potential of the native and modified CNF samples, suspended in different media (water and mixture water-ethanol/acetone with various content of organic solvents), were assessed by dynamic light scattering (DLS) using Zetasizer, Nano ZS ZEN360 (Malvern Instruments Ltd., UK) at 25 ± 0.1°C, and the DTS1070 disposable folded capillary cell. The analysis was performed by applying the following parameters: refractive index of cellulose (1.47), refractive indexes of solvents (1.33, 1.363, and 1.357, for water, ethanol, and acetone, respectively), and viscosities (0.8872, 1.1734, and 0.3084 cP for water, ethanol, and acetone, respectively). A field of 150 V was applied across the nominal electrode spacing of 16 mm. The samples were prepared at concentrations of 0.01 wt.-% and measured at around pH 7 after dispersing them at 10.000 rpm for 3 min using Ultraturax IKA GmbH (Germany). The presented values are average from at least two individual measurements. 2.4. Evaluation of the antimicrobial ability of NH 2 − CNFs and Ag/NH 2 − CNFs The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CNF suspensions were provided by the National Laboratory of Health, Environment, and Food (NLZOH), Maribor, Slovenia, according to the standard test method E2149-10 using Gram-negative bacteria Escherichia coli ( E. coli , DSM 1576) and Pseudomonas aeruginosa ( P. aeruginosa , DSM 1128 ) , Gram-positive bacterium Staphylococcus aureus ( S. aureus , DSM 799), and fungus Candida albicans ( C albicans , DSM 1386) as testing microorganisms. Briefly, NH 2 -CNF and Ag/NH 2 -CNF suspensions (1.55 and 2.4 wt.-%, respectively) were added to a 0.5 mL McFarland standard broth for one hour. 0.1. mL bacteria cell suspensions, in the concentration range of 1.0×10 7 -1.0×10 8 Colony-Forming Units (CFU/mL) and fungal cell suspension of 1.5×10 5 CFU/mL were prepared and added to the CNF suspensions. The control experiments, i.e., experiments without CNF samples in the microorganism culture, were performed similarly. After the 24-hour contact, samples were submitted to serial dilutions and plated in Petri dish plates in triplicates. Microorganisms' growth was followed by tube opacity, and dilution when the microbial growth is not detectable is considered the MIC value. The microbial growth in the absence of CNF samples was further evaluated on Mueller Hinton plates from tubes without noticeable microbial growth, and the MBC value is the concentration when the microbial growth on the solid medium does not occur. 3. Results and Discussion The synthesis of inorganic-organic hybrid particles consisting of Ag NPs and amino-functionalized cellulose nanofibrils (NH 2 − CNFs) is a two-step process (Scheme 1 ). In the first step, NH 2 − CNFs were prepared using a straight-forward procedure based on CNFs oxidation by sodium periodate (NaIO 4 ) to obtain dialdehyde nanocellulose followed by a reaction with ethylenediamine (C 2 H 4 (NH 2 ) 2 ) through imine intermediate by sodium borohydride (NaBH 4 ) that provide the final product, NH 2 − CNFs [ 32 , 33 ]. The high content of free amino groups, estimated to be around 5.0 mM/g, is a significant feature of NH 2 − CNFs. The amino groups are reactive and capable of reducing silver ions to metallic silver, free [ 23 , 24 ], or attached to inorganic [ 28 , 29 ] or organic [ 25 – 27 ] supports. So, in the second synthetic step (Scheme 1 ), Ag NPs were in-situ prepared by the electron transfer reaction from the amino groups and their transformation into imino groups, and thus conjugated to CNFs over the lone electron pairs of the N atoms from amino groups [ 24 , 28 , 29 ]. The abundant presence of free amino groups in NH 2 − CNFs (Table 1 ) provides the possibility to prepare inorganic-organic hybrid micro-large particles with a high content of inorganic phase. Accordingly, the equimolar ratio between free amino groups and Ag + ions was used to prepare the Ag/NH 2 − CNFs hybrid particles with potential application for wastewater disinfection. Table 1 The hydrodynamic size, ζ-potential, surface charge at pH 7, and a total charge of native CNFs, NH 2 − CNFs, and Ag/NH 2 − CNFs suspended in miliQ water. Sample Size at pH 7 (µm) ζ-potential at pH 7 (mV) Charge at pH 7 (mmol/g) Total charge (mmol/g) CNFs 4.2±1.0 -31.6 ± 1.0 0.14 ± 0.01 0.12 ± 0.01 NH 2 − CNFs 10.5±5.6 + 31.3 ± 1.9 2.44 ± 0.04 5.99 ± 0.04 Ag/NH 2 − CNFs 27.7±9.6 + 19.9 ± 4.7 1.00 ± 0.01 4.64 ± 0.04 Before antimicrobial tests, the samples obtained in each synthetic step were thoroughly characterized. The coloration of NH 2 − CNFs support indicates the formation of Ag NPs, and the optical changes induced by the appearance of Ag NPs are shown in Fig. 1 A. Opposite to NH 2 − CNFs support, the Ag/NH 2 − CNFs hybrid absorbs in the visible and near-infrared spectral region with surface plasmon resonance bands peaking around 400 and 550 nm, indicating a relatively small size of metallic silver. The peak at around 400 nm is not well-resolved due to overlap with the absorption of NH 2 − CNFs support. The TG measurement in the air was applied to evaluate the content of the inorganic phase in the hybrid (Fig. 1 B). The residual mass of CNFs and NH 2 − CNFs samples at high temperatures (> 700 ºC) was negligible. On the other hand, the residual mass of the Ag/NH 2 − CNFs, corresponding to the content of metallic silver, was around 20 wt.-%. Based on the concentration of precursors, free amino groups in NH 2 − CNFs, and Ag + ions, the calculated maximal content of metallic silver is 35 wt.-%. The experimentally measured lower inorganic phase content is the most likely consequence of amino groups' steric hindrance during the Ag NPs formation. The wide-angle XRD pattern of the Ag/NH 2 − CNFs hybrid, presented in Fig. 2 , gave diffraction peaks at 38.1, 43.7, 64.3, and 77.3º belonging to the (111), (200), (220), and (311) crystal planes of face-centered-cubic silver (Card No: 9013050). The average crystallite size of silver was estimated to be around 5 nm based on the half-width of diffraction peaks and using Scherrer’s equation. The XRD spectra of CNFs and NH 2 − CNFs, shown in the inset of Fig. 2 , correlated with the literature data [ 34 ], exhibiting the principal peaks around 16.5, 22.5, and 35° which is attributed to the overlapping (1–10) and (110), (200) and (004) planes of monoclinic cellulose, respectively. The empirical equation was applied to calculate the crystallinity index (CI) of CNFs and NH 2 − CNFs using the intensity of the highest XRD peak (I 002 ) and the minimum intensity at a position between the (002) and the (101) peaks (I AM ), which was at about 18.3° [ 35 ]: $$\text{C}\text{I}= \frac{{\text{I}}_{002}-{\text{I}}_{\text{A}\text{M}}}{{\text{I}}_{002}} \times 100\text{\%}$$ 1 The crystallinity indexes of CNFs and NH 2 − CNFs were found to be around 58.3 and 32.7%, indicating that the functionalization steps (above all, the oxidation phase) slightly influenced the crystal-like arrangement of hydrogen-bonded CNFs and consequently their morphology [ 36 ]. The thorough morphological characterization of the Ag/NH 2 − CNFs composite was performed using TEM. Low-magnification TEM images of Ag/NH 2 − CNFs composite (Fig. 3 , A and B) indicated the presence of randomly distributed spherical nanometer in size (< 10 nm) Ag NPs, although agglomeration is seldom noticeable. So, there was a good agreement concerning the size estimation of Ag NPs between TEM, XRD, and spectroscopy data. Analysis of the selected area electron diffraction (SAED) pattern (Fig. 3 C) revealed the presence of diffraction rings consistent with the inverse face-centered-cubic crystalline silver structure, and the analysis of the EDX spectrum (Fig. 3 D) showed the presence of a pronounced peak corresponding to silver. Finally, a high-resolution TEM image displays typical Ag NPs attached to NH 2 − CNFs support (Fig. 3 E). The morphological changes occurring in each step of the amino-functionalization process of CNFs, including the influence of dispersion media, were in detail described in our recent publications [ 31 , 32 ] and are omitted in the present study. It is well-known that the surface charge of Gram-positive and Gram-negative bacteria is negative in media of different acidities and ionic strengths [ 37 ]. Representative of Gram-negative bacteria, E. coli , has a more negatively charged cell wall than typical Gram-positive bacteria, S. aureus . So, it is crucial to determine the pH-dependent surface charge and ζ-potential of all samples (CNFs, NH 2 − CNFs, and Ag/NH 2 − CNFs) from the electrostatic interaction point of view. Also, the potentiometric titration curves (Fig. 4 ) and the pH-dependent ζ-potential values (Fig. 5 A) indicated chemical changes occurring during the synthetic route from CNFs over NH 2 − CNFs to Ag/NH 2 − CNFs. The native CNFs are negatively charged in the entire pH region, showing a negligible bend of negative charge (0.12 mmol/g) at pH around 4–5, related to the seldom presence of anionic surface groups, preferably carboxylic, formed during the preparation of CNFs or due to residual lignin, responsible for ζ-potential of around − 31.6 mV at pH 7 (see Table 1 ). The titration curves (forth and back) of pure HMDA (inset in Fig. 4 ) show a steady high positive charge in a broad pH range, up to the pH 10, close to its deprotonation constant (pK = 11; [ 38 ]) where a steep decrease takes place. So, the functionalization of CNFs with HMDA, followed by the introduction of amino groups, leads to positively surface-charged NH 2 − CNFs (Fig. 4 ) at pH values lower than 9. Also, the titration curves of the NH 2 − CNFs have different shapes compared to the native HMDA. The gradual increase of positive charge is noticeable by increasing the acidity with a barely noticeable bend in the intermediate pH range, confirming the consumption of one of the amino groups from HMDA during the functionalization of CNFs and yielding a total charge of around 5.99 mmol/g. In particular, at pH 7, where the material property is the most important from the applicative side, the ζ-potentials of the NH 2 − CNFs is + 31.3 mV with 2.44 mmol/g of functional groups. These results agree with our recently published data [ 31 , 32 ]. The formation of Ag NPs and their conjugation to functionalized CNFs over amino groups follows a significant decrease in surface charge and ζ-potential (Fig. 4 and Fig. 5 A, respectively). Consequently, the total charge is about 4.64 mmol/g, and at pH 7, ζ-potential is + 19.9 mV with corresponding 1.0 mmol/g functional groups (Table 1 ). The presence of remaining free amino groups in Ag/NH 2 − CNFs indicates non-stoichiometrical conjugation of Ag NP to NH 2 -CNF, and it can be related to the formation mechanism of metallic silver rather than individual conjugation. The pH-dependent potentiometric titration and ζ-potential measurements are in agreement with the thermogravimetric estimation of the content of silver in Ag/NH 2 − CNFs, found to be lower than expected for the stoichiometric reaction between Ag + ions and amino groups. In addition, the ζ-potentials and the average size of NH 2 − CNFs and Ag/NH 2 − CNFs were thoroughly analyzed in mixed solvents, water-ethanol or water-acetone of different compositions, at pH 7, and compared with results obtained in water (Fig. 5 B). It was confirmed that the presence of hydrophobic amino-bearing molecules (HMDA contains ethyl units) attached to CNFs induced their aggregation in water, which can be reduced in the presence of highly polar solvents [ 31 , 32 ]. First, the hydrodynamic size analysis of the Ag/NH 2 − CNFs dispersed in water revealed the presence of significantly larger micro-sized particulates (27.7 µm) compared to the NH 2 − CNFs with high polydispersity and average size of 10.5 µm. Generally, the formation of several micrometers in size NH 2 − CNFs is related to a both-sides (crosslinking) attachment of HMDA to pre-oxidized CNFs that also occurred, besides one-side (grafting) reaction, as well as the formation of aggregates due presence of hydrophobic methylene chain in attached HMDA, as already established in our previous studies [ 31 , 32 ]. On the other hand, a diminished ζ-potential upon the attachment of Ag NPs to the NH 2 − CNFs support, from + 31.3 to + 19.9 mV (Table 1 and Fig. 5 B), i.e., the decrease of surface charge (Fig. 4 ) is responsible for the formation of significantly larger Ag/NH 2 − CNFs hybrids. However, the differences in the ζ-potentials between NH 2 − CNFs and Ag/NH 2 − CNFs are almost negligible for any composition of studied mixed solvents. For example, ζ-potentials of 4.7–3.5 µm large NH 2 − CNFs in miliQ water containing 12, 25, and 50 vol.-% of ethanol are about + 20.0, + 27.0, and + 31.5 mV, respectively, while corresponding values for the 5.9–5.3 µm large Ag/NH 2 − CNFs are + 23.8, + 28.1, and + 28.5 mV, respectively. As a consequence of the similar ζ-potentials in mixed solvents, the hydrodynamic sizes of NH 2 − CNFs and Ag/NH 2 − CNFs, determined by the DLS, are close to each other, within experimental errors, significantly smaller compared to being dispersed in water, and in all cases around 5 µm. Opposite to the mixed water-ethanol solvent, the increase of volume percent of aprotic acetone (does not contain O − H group available for H bonding) in water leads to a decrease of ζ-potentials from around + 36 to + 19 mV of both NH 2 − CNFs and Ag/NH 2 − CNFs. The hydrodynamic size of NH 2 − CNFs and Ag/NH 2 − CNFs is less than 5 µm, except for the mixture with the highest content of acetone (50 vol.-%), where both samples have the lowest ζ-potentials (around + 20 mV). To evaluate the antimicrobial ability of prepared Ag/NH 2 − CNFs, minimum inhibition concentrations (MIC) and minimum bactericidal concentrations (MBC) were measured against Gram-positive bacteria S. aureus , Gram-negative bacteria E. coli and P. aeruginosa , and fungi C. albicans , and compared with the antimicrobial ability of NH 2 − CNFs. This dilution method provides concentration-dependent antimicrobial activity measurements of prepared samples diluted two times up to 128 times. The concentrations of NH 2 − CNFs and Ag/NH 2 − CNFs stock solutions were 15.5 and 24.0 mg/mL, respectively, and the content of silver in the Ag/NH 2 − CNFs was around 20 wt.-%. The CNFs have no inherent antimicrobial activity [ 32 , 39 ], so the control sample did not show any antimicrobial activity. As expected, the NH 2 − CNFs display growth inhibition of all studied microbial species. The MIC values obtained in this study with published MIC values in our previous studies [ 31 , 32 ] are presented in Table 2 . Besides the hydrophobic tail, the presence of the protonated, positively charged amino groups is essential for the efficient antimicrobial activity of NH 2 − CNFs since both the hydrophobic and electrostatic interaction with the hydrophobic and negatively charged cell walls leads to increased contact with microorganisms [ 40 ], compromising its integrity and leading to leakage of cytoplasmic content and, ultimately, cell lysis, thereby resulting in a bactericidal effect [ 21 ]. The differences in MIC values of NH 2 − CNFs, particularly for Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli , are the most likely consequence of the different testing methodologies causing differences in the dispersibility of samples. Table 2 Minimum inhibition concentrations (MIC) and minimum bactericidal concentrations (MBC) of NH 2 − CNFs and Ag/NH 2 − CNFs. NH 2 − CNFs Ag/NH 2 − CNFs MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL) S. aureos 0.96 ~ 3 * 0.12 ** 4 * 1.9 + E. coli 0.12 8 * 1.9 ** 16 * 3.0 12 P. aeruginosa 0.48 n.d. nd n.d. 3.0 12 C. albicans 0.24 n.d. 0.24 ** n.d. 3.0 min after 24 h * Data from reference 32. ** Data from reference 31. nd ‒ not determined Besides MIC, the MBC values for NH 2 − CNFs and Ag/NH 2 − CNFs are also collected in Table 2 , and based on these data, we can conclude the following. First, both composite display growth inhibition of all studied microbial species. On the other hand, the microorganism-killing activity that prevents their colonization occurs either at higher silver concentrations than inhibition or does not take place in the case of S. aureus in the studied concentration range. Second, the MIC values are higher than the ones obtained for free-standing similar in-size Ag NPs [ 41 ] or immobilized Ag NPs on glass support [ 42 ]. However, MIC and MBC values depend on the morphology (size and shape), preparation method, and surface properties of Ag NPs. So, a similar MIC value against S. aureus (0.625 mg/mL) is reported by Parvekar et al. [ 43 ] for 5 nm in size Ag NPs, while Ayala-Núñez et al. [ 44 ] demonstrated that MIC and MBC of 10 nm Ag NPs are in concentrations of 1.35 mg/mL against S. aureus . Third, the MIC values obtained for Ag/NH 2 − CNFs are lower than those for NH 2 − CNFs. Although free amino groups are present in the Ag/NH 2 − CNFs, most likely due to their steric hindrance, the mechanism of the antimicrobial action of the Ag/NH 2 − CNFs is different, switching to the antimicrobial activity of Ag NPs. The antimicrobial action of immobilized Ag NPs can be due to direct contact of microbial species with Ag NPs, immobilized or detached in solution, and with released Ag + ions in solution [ 45 ]. The advantage of immobilized over colloidal Ag NPs is, as shown by Lv et al. [ 46 ], their higher stability and consequently suppressed oxidation that provides long-term antimicrobial activity. From the practical point of view, small and controlled release of Ag NPs and Ag + ions in wastewater is the essential prerequisite for its disinfection. So, we aged the Ag/NH 2 − CNFs composite in water and, after one month, applied the ICP-AES technique to determine the concentration of silver in the supernatant. The ICP-AES measurement revealed that the total concentration of released silver is less than 1% of the content of Ag NPs in Ag/NH 2 − CNFs composite. The ICP-AES technique does not distinguish the valence state of silver, and since the supernatant was yellowish, the presence of detached Ag NP from the composite in the supernatant was proven by spectroscopy measurements. The absorption spectrum of the supernatant (Supporting Information, Figure S1 ) has the surface plasmon resonance band peaking at 410 nm, characteristic of nanometer-in-size Ag particles. Nevertheless, although the Ag/NH 2 -CNF composite serves as a reservoir of silver, the antimicrobial activity will be present even after the complete release of silver since the NH 2 − CNFs can capture and deactivate the bacteria from wastewater [ 47 ]. It is well-known that released silver in any form (ionic or metallic) retains its cytotoxicity and ecotoxicity even at a concentration as low as 1.0 mg/L [ 41 , 48 ]. So, a trade-off between release rate and efficiency of the antimicrobial action is a prerequisite for the Ag NPs application as a disinfection agent. To conclude, considering the high content of Ag NPs in the Ag/NH 2 − CNFs (20 wt.-%), slow release of Ag + ions to surrounding media, and negligible detachment of Ag NPs, it seems that the prepared sample is suitable for long-term antimicrobial action and worth further investigating its potential application. Conclusion The free amino groups in NH 2 − CNFs provided a simple way to prepare inorganic-organic hybrid particles with a high content of Ag NPs (20 wt.-%). Based on the characterization and antimicrobial activities of prepared samples, several conclusions can be drawn. Incorporating a nanometer large Ag NPs does not significantly affect the hydrodynamic size, ζ-potential, and surface charge of NH 2 − CNFs, i.e., hybrid retained desirable properties of its organic component. The inorganic-organic (Ag/NH 2 − CNFs) hybrid and NH 2 − CNFs display similar antimicrobial activity, although the mechanism of their antimicrobial action is different, i.e., electrostatic interactions via amino groups versus Ag + ions released from Ag NPs. Slow release of Ag ions and negligible detachment of Ag NPs to surrounding media, i.e., stability of prepared hybrid, ensure its long-term antimicrobial action. We believe that the obtained results are a good starting point to extend further investigations towards potential applications of hybrid consisting of NH 2 − CNFs and Ag/NH 2 − CNFs for wastewater treatment on a large scale. Declarations Funding This research has received funding from the Slovenian Research and Innovation Agency (Research Programme P2-0424, Research projects BI-RS 18/19-043). Also, this research was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract Number: 451-03-66/2024-03/200017). Acknowl edgmen t The authors are grateful to Vera Vivod, MSc., for preparing the samples and performing the zeta-size analysis. Data availability The authors declare that all the data generated or analyzed during this study are available within the article. Author contributions Vesna Lazić: Investigation, Data curation, Methodology, Funding acquisition. Jovan M. Nedeljković: Conceptualisation, Methodology, Investigation, Visualisation, Writing – original draft, Writing – review and editing. Vanja Kokol: Conceptualisation, Funding acquisition, Methodology, Investigation, Visualisation, Writing – original draft, Writing – review and editing. Ethical approval Not applicable. Consent for participate All of the authors consent to participate in this manuscript. Consent to publish All of the authors consent to publish this manuscript Research involving human participants or animals Not applicable. Declaration of conflicting interests The authors declared no conflicts of interest. References D. Trache, A.F. Tarchoun, M. Derradji, T.S. Hamidon, N. Masruchin, N. Brosse, M.H. Hussin, Nanocellulose: From Fundamentals to Advanced Applications, Front. Chem., 8, 392 (2020). B. Thomas, M.C. Raj, J. Joy, A. Moores, G.L. Drisko, C. Sanchez, Nanocellulose, a versatile green platform: From biosources to materials and their applications, Chem. Rev., 118, 11575–11625 (2018). P. Thomas, T. Duolikun, N.P. Rumjit, S. Moosavi, C.W. Lai, M.R.B. Johan, L.B. Fen, Comprehensive review on nanocellulose: Recent developments, challenges and future prospects, J. Mech. Behav. Biomed., 110, 103884 (2020). G. Dalei, S. Das, M. Pradhan, Dialdehyde cellulose as a niche material for versatile applications: An overview, Cellulose, 29, 5429–5461 (2022). V. Thakur, A. Guleria, S. Kumar, S. Sharma, K. Singh, Recent advances in nanocellulose processing, functionalization and applications: A review, Mater. Adv., 2, 1872–1895 (2021). V. Ilić, Z. Šaponjić, V. Vodnik, B. Potkonjak, P. Jovančić, J. Nedeljković, M. Radetić, The influence of silver content on antimicrobial activity and color of cotton fabrics functionalized with Ag nanoparticles, Carbohyd. Polym., 78, 564–569 (2009). B. Jia, Y. Mei, L. Cheng, J. Zhou, L. Zhang, Preparation of copper nanoparticles coated cellulose films with antibacterial properties through one-step reduction, ACS Appl. Mater. Inter., 4, 2897–2902 (2012). Y. Xu, S. Li, X. Yue, W. Lu, Review of silver nanoparticles (AgNPs)-cellulose antibacterial composites, BioRes., 13, 2150–2170 (2018). D. Mihailović, Z. Šaponjić, M. Radoičić, S. Lazović, C.J. Baily, P. Jovančić, J. Nedeljković, M. Radetić, Functionalization of cotton fabrics with corona/air RF plasma and colloidal TiO 2 nanoparticles, Cellulose, 18, 811–825 (2011). B.L. Carpenter, F. Scholle, H. Sadeghifar, A.J. Francis, J. Boltersdorf, W.W. Weare, D.S. Argyropoulos, P.A. Maggard, R.A. Ghiladi, Synthesis, characterization, and antimicrobial efficacy of photomicrobicidal cellulose paper, Biomacromolecules, 16, 2482–2492 (2015). A. Yoosefi Booshehri, R. Wang, R. Xu, Simple method of deposition of CuO nanoparticles on a cellulose paper and its antibacterial activity, Chem. Eng. J., 262, 999–1008 (2015). K. Lefatshe, C.M. Muiva, L.P. Kebaabetswe, Extraction of nanocellulose and in-situ casting of ZnO/cellulose nanocomposite with enhanced photocatalytic and antibacterial activity, Carbohyd. Polym., 164, 301–308 (2017). S.-W. Zhao, C.-R. Guo, Y.-Z. Hu, Y.-R. Guo, Q.-J. Pan, The preparation and antibacterial activity of cellulose/ZnO composite: a review, Open Chem., 16, 9–20 (2018). T.C. Mokhena, M.J. John, Cellulose nanomaterials: New generation materials for solving global issues, Cellulose, 27, 1149–1194 (2020). Gülsu, E. Yüksektepe, Preparation of gentamicin conjugated cellulose nanocrystals and evaluation of efficacy on different microorganisms, Eur. J. Sci. Tech., 27, 1105–1112 (2021). C. Amara, A. El Mahdi, R. Medimagh, K. Khwaldia, Nanocellulose-based composites for packaging applications. Curr. Opin. Green Sust., 31, 100512 (2021). L. Zhang, P. Yan, Y. Li, X. He, Y. Dai, Z. Tan, Preparation and antibacterial activity of a cellulose-based schiff base derived from dialdehyde cellulose and L-lysine, Ind. Crop Prod., 145, 112126 (2020). X. He, Y. Li, L. Zhang, R. Du, Y. Dai, Z. Tan, Preparation of 2,3-dialdehyde microcrystalline cellulose particles crosslinked with ε-poly-L-lysine and their antibacterial activity, Cellulose, 28, 2833–2847 (2021). G. Dalei, S. Das, M. Pradhan, Dialdehyde cellulose as a niche material for versatile applications: An overview, Cellulose, 29, 5429–5461 (2022). L. Timofeeva, N. Kleshcheva, Antimicrobial polymers: Mechanism of action, factors of activity, and applications, Appl. Microbiol. Biot., 89, 475–492 (2011). O.M. Bondarenko, M. Heinlaan, M. Sihtmäe, A. Ivask, I. Kurvet, E. Joonas, A. Jemec, M. Mannerström, T. Heinonen, R. Rekulapelly, S. Singh, J. Zou, I. Pyykkö, D. Drobne, A. Kahru, Multilaboratory evaluation of 15 bioassays for (eco) toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID, Nanotoxicology, 10, 1229–1242 (2016). M. Tavakolian, S.M. Jafari, T.G. van de Ven, A review on surface-functionalized cellulosic nanostructures as biocompatible antibacterial materials, Nano-Micro Lett., 12, 1–23 (2020). M. Chen, Y.G. Feng, X. Wang, T.C. Li, J.Y. Zhang, D.J. Qian, Silver nanoparticles capped by oleylamine: formation, growth, and self-organization, Langmuir, 23, 5296–5304 (2007). S. Mourdikoudis, L.M. Liz-Marzan, Oleylamine in Nanoparticle Synthesis, Chem. Mater., 25, 1465–1476 (2013). I.D. Vukoje, E.S. Džunuzović, V.V. Vodnik, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Synthesis, characterization, and antimicrobial activity of poly(GMA-co-EGDMA) polymer decorated with silver nanoparticles, J. Mater. Sci., 49, 6838–6844 (2014). I.D. Vukoje, E.S. Džunuzović, D.R. Lončarević, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Synthesis, characterization, and antimicrobial activity of silver nanoparticles on poly(GMA-co-EGDMA) polymer support, Polym. Composite, 38, 1206–1214 (2017). I.D. Vukoje, E.S. Džunuzović, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Size-Dependent Antibacterial Properties of Ag Nanoparticles Supported by Amino-Functionalized Poly(GMA-co-EGDMA) Polymer, Polym. Composite, 40, 2901–2907 (2019). V. Lazić, I. Smičiklas, J. Marković, D. Lončarević, J. Dostanić, S.P. Ahrenkiel, J.M. Nedeljković, Antibacterial ability of supported silver nanoparticles by functionalized hydroxyapatite with 5-aminosalicylic acid, Vacuum, 148, 62–68 (2018). V. Lazić, K. Mihajlovski, A. Mraković, E. Illés, M. Stoiljković, S.P. Ahrenkiel, J.M. Nedeljković, Antimicrobial activity of silver nanoparticles supported by magnetite, ChemistrySelect, 4, 4018–4024 (2019). V. Lazić, V. Vivod, Z. Peršin, M. Stoiljković, I.S. Ratnayake, P.S. Ahrenkiel, J.M. Nedeljković, V. Kokol, Dextran-coated silver nanoparticles for improved barrier and controlled antimicrobial properties of nanocellulose films used in food packaging, Food Packaging Shelf, 26, 100575 (2020). V. Kokol, V. Vivod, Z. Peršin Fratnik, M. Čolić, M. Kolar, Antimicrobial properties of viscose yarns ring-spun with integrated amino-functionalized nanocellulose, Cellulose, 28, 6545–6565 (2021). V. Kokol, S. Novak, V. Kononenko, M. Kos, V. Vivod, N. Gunde-Cimerman, D. Drobne, Antibacterial and degradation properties of dialdehyded and aminohexamethylated nanocelluloses, Carbohyd. Polym., 311, 120603–120619 (2023). Q. Xu, X. Huang, L. Guo, Y. Wang, L. Jin, Enhancing Removal of Cr(VI), Pb 2+ , and Cu 2+ from Aqueous Solutions Using Amino-Functionalized Cellulose Nanocrystal, Molecules, 26, 7315 (2021). M.L. Tummino, E. Laurenti, P. Bracco, C. Cecone, V. La Parola, C. Vineis. M.L. Testa, Antibacterial properties of functionalized cellulose extracted from deproteinized soybean hulls, Cellulose, 30, 7805–7824 (2023). S. Park, J.O. Baker, M.E Himmel, P.A. Parilla, D.K. Johnson, Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance, Biotechnol. Biofuels, 3:10 (2010). A. Errokh, A. Magnin, J.-L. Putaux, S. Boufi, Morphology of the nanocellulose produced by periodate oxidation and reductive treatment of cellulose fibers, Cellulose, 25, 3899–3911 (2018). R. Sonohara, N. Muramatsu, H. Ohshima, T. Kondo, Difference in surface properties between Escherichia coli and Staphylococcus aureus as revealed by electrophoretic mobility measurements, Biophys. Chem., 55, 273–277 (1995). B.K. Mondal, S.S. Bandyopadhyay, A.N. Samanta, Kinetics of CO 2 absorption in aqueous hexamethylenediamine, Int. J. Greenhouse Gas Control, 56, 116–125 (2017). S. Rashki, N. Shakour, Z. Yousefi, M. Rezaei, M. Homayoonfal, E. Khabazian, F. Atyabi, F. Aslanbeigi, R.S. Lapavandani, S. Mazaheri, M.R. Hamblin, H. Mirzaei, Cellulose-Based Nanofibril Composite Materials as a New Approach to Fight Bacterial Infections, Front. Bioeng. Biotech., 9, 732461 (2021). M. Rajagopal, S. Walker, Envelope structures of gram-positive bacteria, Curr. Top. Microbiol. Immunol., 404, 1–44. (2017). A. Panáček, L. Kvítek, R. Prucek, M. Kolář, R. Večeřová, N. Pizúrová, V.K. Sharma, T. Nevěčná, R. Zbořil, Silver Colloid Nanoparticles: Synthesis, Characterization, and Their Antibacterial Activity, J. Phys. Chem. B, 110,16248–16253 (2006). S. Agnihotri, S. Mukherji, S. Mukherji, Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial Efficacy, RSC Adv., 4, 3974–3983 (2014). P. Parvekar, J. Palaskar, S. Metgud, R. Maria, S. Dutta, The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against Staphylococcus aureus , Biomater. Investig. Dent., 7, 105–109 (2020). N.V. Ayala-Núñez, H.H. Lara Villegas, L. del Carmen Ixtepan Turrent, C. Rodríguez Padilla, Silver Nanoparticles Toxicity and Bactericidal Effect Against Methicillin-Resistant Staphylococcus aureus: Nanoscale Does Matter, NanoBiotechnology, 5, 2–9 (2009). S. Agnihotri, S. Mukherji, S. Mukherji, Immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver, Nanoscale, 5, 7328–7340 (2013). M. Lv, S. Su, Y. He, Q. Huang, W. Hu, D. Li, C. Fan, S.-T. Lee, Long-Term Antimicrobial Effect of Silicon Nanowires Decorated with Silver Nanoparticles, Adv. Mater., 22, 5463–5467 (2010). V. Kokol, M. Kos, V. Vivod, N. Gunde-Cimerman, Cationised Fibre-Based Cellulose Multi-Layer Membranes for Sterile and High-Flow Bacteria Retention and Inactivation, Membranes,13, 284 (2023). A. Panáček, M. Kolář, R. Večeřová, R. Prucek, J. Soukupová, V. Kryštof, P. Hamal, R. Zbořil, L. Kvítek, Antifungal activity of silver nanoparticles against Candida spp., Biomaterials, 30, 6333–6340 (2009). Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Schema.png Scheme 1. Synthetic pathway for preparation of inorganic-organic hybrid consisting of Ag NPs and amino-functionalized CNFs. SupportingInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4507463","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314630116,"identity":"58ec612d-17cc-44ea-8d06-b8d2bb482194","order_by":0,"name":"Vesna Lazic","email":"","orcid":"","institution":"Vinča Institute of Nuclear Sciences − National Institute of the Republic of Serbia, University of Belgrade, Centre of Excellence for Photoconversion","correspondingAuthor":false,"prefix":"","firstName":"Vesna","middleName":"","lastName":"Lazic","suffix":""},{"id":314630117,"identity":"c709d23d-88cd-47af-a121-e2f15c041a9a","order_by":1,"name":"Jovan M. Nedeljković","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACxgYQyWbDwMYAQiRoSSNBCwSwHQaTxClmbu8xe/Ch7Hxin3QD28MvDHbyDNLHH+B3WM8Zc8MZ524ntskcYDeWYUg2bODLMcCvZUZamjRv221jNokENmkJBuYEBh4e/A4Da/nbdg6mpR6ohZ2Aw2YkH5NmbDsgB9Ii+YHhMFALAwGH9Rw+JtlzLhmoJbHdmMHguGEbDw9+LYbtjW0SP8rseOSB1j38UVEtz0/IYYYNCAsbmEHmE4wdeRRX/iCkfBSMglEwCkYkAABIjjiIlAWNKAAAAABJRU5ErkJggg==","orcid":"","institution":"Vinča Institute of Nuclear Sciences − National Institute of the Republic of Serbia, University of Belgrade, Centre of Excellence for Photoconversion","correspondingAuthor":true,"prefix":"","firstName":"Jovan","middleName":"M.","lastName":"Nedeljković","suffix":""},{"id":314630118,"identity":"7f96c41f-83a9-4c29-aa42-e92ce49e6a4b","order_by":2,"name":"Vanja Kokol","email":"","orcid":"","institution":"Univeristy of Maribor, Faculty of Mechanical Engineering","correspondingAuthor":false,"prefix":"","firstName":"Vanja","middleName":"","lastName":"Kokol","suffix":""}],"badges":[],"createdAt":"2024-05-31 08:36:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4507463/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4507463/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59176260,"identity":"cb7563e6-7c94-4708-be55-467606454d8b","added_by":"auto","created_at":"2024-06-27 09:37:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2141759,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Kubelka-Munk transformations of reflection data of NH\u003csub\u003e2\u003c/sub\u003e−CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e−CNFs. (\u003cstrong\u003eB\u003c/strong\u003e) TG curves, measured in the air at a heating rate of 10 ºC/min, of CNFs, NH\u003csub\u003e2\u003c/sub\u003e−CNFs, and Ag/NH\u003csub\u003e2\u003c/sub\u003e−CNFs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/7997774f46513719cc9e8f97.png"},{"id":59176854,"identity":"5d0f8b80-718c-427a-b7c4-80bac003d215","added_by":"auto","created_at":"2024-06-27 09:45:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":242524,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD pattern of the Ag/NH\u003csub\u003e2\u003c/sub\u003e−CNFs hybrid; inset: the XRD patterns of CNFs and NH\u003csub\u003e2\u003c/sub\u003e−CNFs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/90695cfb1fe47960edf696d1.png"},{"id":59177463,"identity":"8baa0817-421f-415a-ac82-543cd24d8f76","added_by":"auto","created_at":"2024-06-27 09:53:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3214393,"visible":true,"origin":"","legend":"\u003cp\u003eLow-magnification TEM images of Ag/NH\u003csub\u003e2\u003c/sub\u003e−CNFs composite (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e) and corresponding SAED pattern (\u003cstrong\u003eC\u003c/strong\u003e) as well as EDX spectrum (\u003cstrong\u003eD\u003c/strong\u003e). High-magnification TEM image of typical Ag NP attached to NH\u003csub\u003e2\u003c/sub\u003e−CNFs support (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/e0d76bdaafbd26fec5c7268d.png"},{"id":59176263,"identity":"3b2fd268-9403-4aef-81b8-2814f5eeeec2","added_by":"auto","created_at":"2024-06-27 09:37:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":279829,"visible":true,"origin":"","legend":"\u003cp\u003ePotentiometric titration curves of native CNFs, NH\u003csub\u003e2\u003c/sub\u003e-CNF, and Ag/NH\u003csub\u003e2\u003c/sub\u003e-CNF with corresponding charges suspended in water; concentration: 0.001 wt.-%.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/be6ac44e8285f9a15f61bc93.png"},{"id":59176261,"identity":"fdb4a32f-afdd-43e7-9d61-c06330afe78b","added_by":"auto","created_at":"2024-06-27 09:37:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298431,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) ζ-potentials and (\u003cstrong\u003eB\u003c/strong\u003e) average hydrodynamic size distribution of native CNFs, NH\u003csub\u003e2\u003c/sub\u003e-CNF, and Ag/NH\u003csub\u003e2\u003c/sub\u003e-CNF in water and water containing different volume percent of ethanol or acetone at pH 7,\u0026nbsp; analyzed by DLS; concentration: 0.001 wt.-%.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/bb8cd61bd7312e1ba0a1e0a7.png"},{"id":60778995,"identity":"03d093f0-73dc-4866-b281-0589ebaa688e","added_by":"auto","created_at":"2024-07-22 00:44:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7635004,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/6e832282-bc37-4f04-8191-374e4df0955d.pdf"},{"id":59177462,"identity":"4abf6951-ea1c-41f6-9983-b67b3d8dd96a","added_by":"auto","created_at":"2024-06-27 09:53:11","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":246208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Synthetic pathway for preparation of inorganic-organic hybrid consisting of Ag NPs and amino-functionalized CNFs.\u003c/p\u003e","description":"","filename":"Schema.png","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/23b52f937fcb1adfe47fce35.png"},{"id":59176266,"identity":"9103bb48-95e8-4285-b431-bfb091bf0813","added_by":"auto","created_at":"2024-06-27 09:37:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":109262,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4507463/v1/912eb5329741c73e32905212.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial activity of amino-modified cellulose nanofibrils decorated with silver nanoparticles","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanocellulose (NC) is one of the most promising eco-friendly, non-toxic materials, obtained from renewable sources (plants, algae, tunicates, and some bacteria), with the potential to replace synthetic polymers in many applications due to their unique properties, including easy processability and the possibility to tailor morphology, specific surface area, crystallinity, and biocompatibility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Because of these unique features, NC is recognized as a suitable material for various current and emerging applications, such as nanocomposites, adhesives, paints, barrier and functional coatings, fibers and filaments, films and foils, membranes and filters, biomedical and pharmaceutical products, supercapacitors, and batteries [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, pristine NC does not display antimicrobial activity, and with the knowledge of the importance of non-toxic biodegradation for the environment, various approaches were proposed to obtain its antimicrobial activity. Generally, two different approach types are recognizable, the first based on surface modification of NC with aldehyde, amino, and quaternary ammonium groups [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and the second based on conjugation with either metal (Au, Ag, and Cu) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], metal oxide nanoparticles (CuO, MgO, ZnO, and TiO\u003csub\u003e2\u003c/sub\u003e) [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], biocidal agents, such as gentamicin [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], or antimicrobial polymers [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFunctionalization of NC with amino groups is of particular interest. On one side, amino-functionalized cellulose nanofibrils (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) display antimicrobial activity due to the electrostatic interaction between positively charged amino groups and negatively charged cell walls that enhanced their contact with microorganisms [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. On the other side, the free amino groups are capable of reducing Ag\u003csup\u003e+\u003c/sup\u003e ions to their metallic form, providing the possibility to prepare free-standing colloidal Ag NPs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] or Ag NPs linked to different types of supports, polymer [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and inorganic [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], with high antimicrobial activity. Besides the antimicrobial activity of Ag NPs on support, when they are easily accessible to microbial species in the surrounding medium, Ag NPs dispersed within a polymer matrix also have antimicrobial activity. Recently, we demonstrated that thin films prepared from cellulose nanofibrils loaded with less than 0.5 wt.-% of a nanometer in size Ag NPs inhibited the growth of \u003cem\u003eE. coli\u003c/em\u003e after five repeated cycles, indicating that might serve as a sustainable replacement for their synthetic counterparts in food packaging applications [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study thus aims to evaluate the antimicrobial activity of hybrid based on amino-functionalized cellulose nanofibrils (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) decorated with Ag NPs, prepared \u003cem\u003ein situ\u003c/em\u003e, taking advantage of the reducing ability of free amino groups. So, combining inorganic (Ag NPs) and organic (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) components into a hybrid (Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs), with the knowledge that each of the components displays antimicrobial activity [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], was intended to improve the antimicrobial performance of hybrid under long time working conditions. Samples were characterized in detail before determination of their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against different microbial species (Gram-negative bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, Gram-positive bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and fungus \u003cem\u003eCandida albicans\u003c/em\u003e). In addition, the content of the inorganic phase was determined by thermogravimetric measurements, an optical property of hybrid originating from Ag NPs was correlated with transmission electron microscopy data, and X-ray diffraction analysis provided the influence of the inorganic component on the crystallinity of NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs. Finally, as a crucial factor influencing the electrostatic interaction of samples with bacteria\u0026rsquo;s wall, pH-dependent total charge and ζ-potentials of samples in different media were determined, including the hydrodynamic size of the hybrids.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe wood-based chain-like cellulose nanofibrils (CNFs) with diameters in the 10\u0026ndash;70 nm range and length of a few micrometers (1\u0026ndash;3 \u0026micro;m) were prepared from bleached softwood pulp, refined in a multistage disc refiner by the University of Maine, The Process Development Center in the USA. Hexamethylenediamine (HMDA, 98% purity) and all other chemicals used were purchased from Sigma-Aldrich Co. Ltd. (USA) and used without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of amino-functionalized CNFs (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) decorated with silver nanoparticles (Ag NPs)\u003c/h2\u003e \u003cp\u003eFirst, the amino-functionalized CNFs (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) were prepared by a two-step reaction, as presented in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The aqueous dispersion of CNFs (1 wt.-%, 100 mL) was first oxidized to exhibit aldehyde functional groups at the C2 and C3 cellulose units positions by mixing it with pre-dissolved sodium periodate (1.6 g NaIO\u003csub\u003e4\u003c/sub\u003e per 1 g of CNF) and stirred for 48 h in the dark at room temperature. The product was washed thoroughly a few times with deionized water for up to 3 days. The functionalization of pre-oxidized CNFs with HMDA was performed through a Schiff-base reaction \u003cem\u003evia\u003c/em\u003e aldehyde groups. For that purpose, 200 ml of 0.5 wt.-% pre-oxidized CNFs suspension was ultrasonicated for 5 min, and then 8 mmol of HMDA was added. The mixture was stirred continuously for 6 h at 30\u0026deg;C, followed by the \u003cem\u003ein-situ\u003c/em\u003e reduction of the resulting imine intermediate at room temperature, employing 0.58 g of NaBH\u003csub\u003e4\u003c/sub\u003e. After stirring for 3 h, the products were washed thoroughly several times with deionized water until a neutral pH was reached and used as prepared.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the next step, the Ag NPs were grown onto NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs by the \u003cem\u003ein-situ\u003c/em\u003e reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions, taking advantage of the reducing ability of free amino groups, which are present in the amino-functionalized CNFs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, the mixture having a 1:1 molar ratio between Ag\u003csup\u003e+\u003c/sup\u003e ions and amino groups (85 mg of AgNO\u003csub\u003e3\u003c/sub\u003e and 2.1 mL of amino-functionalized CNFs in 50 mL of H\u003csub\u003e2\u003c/sub\u003eO) was stirred overnight under reflux at 60\u0026deg;C. The appearance of a yellow-brown color indicated the successful reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions to the metallic silver. Then, the dispersion was washed several times with deionized water and dried at 40\u0026deg;C in a vacuum oven for 24 h. For clarity reasons, Ag NPs conjugated with NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs will be denoted further in the text as Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs\u003c/h2\u003e \u003cp\u003eAbsorption of NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs were evaluated in UV\u0026ndash;Vis\u0026ndash;NIR spectral range by diffuse reflectance measurements (Shimadzu UV\u0026ndash;Visible UV-2600 spectrophotometer equipped with an integrated sphere ISR-2600 Plus).\u003c/p\u003e \u003cp\u003eThe content of metallic silver in Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs was determined by thermogravimetric analysis (TGA) using a Setaram Setsys Evolution-1750 instrument. The TGA measurements were performed under a dynamic air atmosphere (flow rate of 20 cm\u003csup\u003e3\u003c/sup\u003e/min) in the temperature range from room temperature to 1000\u0026deg;C (heating rate of 10\u0026deg;C/min). The content of released silver (Ag\u003csup\u003e+\u003c/sup\u003e ions and Ag NPs) after 30 days of Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs incubation in distilled water at room temperature was determined using inductively coupled plasma optic emission spectroscopy (ICP-OES Thermo Scientific iCAP 7400).\u003c/p\u003e \u003cp\u003eX-ray diffraction (XRD) measurements of prepared samples were carried out using a Rigaku SmartLab instrument with Cu Kα1,2 radiation. The measurements were performed with continuous scanning at 2 \u0026deg;/min and by collecting the data at 0.02\u0026deg; intervals.\u003c/p\u003e \u003cp\u003eMicrostructural characterization of the Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs was performed on a transmission electron microscope (TEM) JEOL JEM-2100 LaB6 operating at 200 kV. TEM images were acquired with a Gatan Orius CCD camera at 2\u0026times; binning.\u003c/p\u003e \u003cp\u003ePotentiometric titrations of native and modified CNF samples suspended in miliQ water were performed to quantify the process-dependent surface charge contributions. The titration was carried out using a dual-burette instrument (Mettler Toledo T-70) equipped with a combined glass electrode (Mettler TDG 117) and filled with 0.1 M HCl (Merck, Titrisol) and 0.1 M KOH (Baker, Dilut-it). Samples soaked in milli-Q water were rinsed in low pH (0.01 M HCl) to convert the basic and acidic groups into protonated forms and then dried at 40\u0026deg;C. The titrations were carried out at room temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C), forward and backward between pH 2 and 11. The molar concentration, related to the overall charge of the side groups, was calculated from the potentiometric titration data. All the reported values are the mean values of duplicate determinations.\u003c/p\u003e \u003cp\u003eThe hydrodynamic size and zeta-potential of the native and modified CNF samples, suspended in different media (water and mixture water-ethanol/acetone with various content of organic solvents), were assessed by dynamic light scattering (DLS) using Zetasizer, Nano ZS ZEN360 (Malvern Instruments Ltd., UK) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C, and the DTS1070 disposable folded capillary cell. The analysis was performed by applying the following parameters: refractive index of cellulose (1.47), refractive indexes of solvents (1.33, 1.363, and 1.357, for water, ethanol, and acetone, respectively), and viscosities (0.8872, 1.1734, and 0.3084 cP for water, ethanol, and acetone, respectively). A field of 150 V was applied across the nominal electrode spacing of 16 mm. The samples were prepared at concentrations of 0.01 wt.-% and measured at around pH 7 after dispersing them at 10.000 rpm for 3 min using Ultraturax IKA GmbH (Germany). The presented values are average from at least two individual measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Evaluation of the antimicrobial ability of NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CNF suspensions were provided by the National Laboratory of Health, Environment, and Food (NLZOH), Maribor, Slovenia, according to the standard test method E2149-10 using Gram-negative bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e, DSM 1576) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e, DSM 1128\u003cem\u003e)\u003c/em\u003e, Gram-positive bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e, DSM 799), and fungus \u003cem\u003eCandida albicans\u003c/em\u003e (\u003cem\u003eC albicans\u003c/em\u003e, DSM 1386) as testing microorganisms.\u003c/p\u003e \u003cp\u003eBriefly, NH\u003csub\u003e2\u003c/sub\u003e-CNF and Ag/NH\u003csub\u003e2\u003c/sub\u003e-CNF suspensions (1.55 and 2.4 wt.-%, respectively) were added to a 0.5 mL McFarland standard broth for one hour. 0.1. mL bacteria cell suspensions, in the concentration range of 1.0\u0026times;10\u003csup\u003e7\u003c/sup\u003e-1.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e Colony-Forming Units (CFU/mL) and fungal cell suspension of 1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL were prepared and added to the CNF suspensions. The control experiments, i.e., experiments without CNF samples in the microorganism culture, were performed similarly. After the 24-hour contact, samples were submitted to serial dilutions and plated in Petri dish plates in triplicates. Microorganisms' growth was followed by tube opacity, and dilution when the microbial growth is not detectable is considered the MIC value.\u003c/p\u003e \u003cp\u003eThe microbial growth in the absence of CNF samples was further evaluated on Mueller Hinton plates from tubes without noticeable microbial growth, and the MBC value is the concentration when the microbial growth on the solid medium does not occur.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe synthesis of inorganic-organic hybrid particles consisting of Ag NPs and amino-functionalized cellulose nanofibrils (NH\u003csub\u003e2\u003c/sub\u003e − CNFs) is a two-step process (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the first step, NH\u003csub\u003e2\u003c/sub\u003e − CNFs were prepared using a straight-forward procedure based on CNFs oxidation by sodium periodate (NaIO\u003csub\u003e4\u003c/sub\u003e) to obtain dialdehyde nanocellulose followed by a reaction with ethylenediamine (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) through imine intermediate by sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e) that provide the final product, NH\u003csub\u003e2\u003c/sub\u003e − CNFs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe high content of free amino groups, estimated to be around 5.0 mM/g, is a significant feature of NH\u003csub\u003e2\u003c/sub\u003e − CNFs. The amino groups are reactive and capable of reducing silver ions to metallic silver, free [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], or attached to inorganic [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] or organic [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] supports. So, in the second synthetic step (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), Ag NPs were \u003cem\u003ein-situ\u003c/em\u003e prepared by the electron transfer reaction from the amino groups and their transformation into imino groups, and thus conjugated to CNFs over the lone electron pairs of the N atoms from amino groups [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The abundant presence of free amino groups in NH\u003csub\u003e2\u003c/sub\u003e − CNFs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) provides the possibility to prepare inorganic-organic hybrid micro-large particles with a high content of inorganic phase. Accordingly, the equimolar ratio between free amino groups and Ag\u003csup\u003e+\u003c/sup\u003e ions was used to prepare the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs hybrid particles with potential application for wastewater disinfection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe hydrodynamic size, ζ-potential, surface charge at pH 7, and a total charge of native CNFs, NH\u003csub\u003e2\u003c/sub\u003e − CNFs, and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs suspended in miliQ water.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize at pH 7\u003c/p\u003e \u003cp\u003e(µm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eζ-potential at pH 7\u003c/p\u003e \u003cp\u003e(mV)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCharge at pH 7\u003c/p\u003e \u003cp\u003e(mmol/g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal charge\u003c/p\u003e \u003cp\u003e(mmol/g)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNFs\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e4.2±1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e-31.6 ± 1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.14 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e0.12 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003e − CNFs\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e10.5±5.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e+ 31.3 ± 1.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e2.44 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e5.99 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAg/NH\u003csub\u003e2\u003c/sub\u003e − CNFs\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e27.7±9.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e+ 19.9 ± 4.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.00 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e4.64 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eBefore antimicrobial tests, the samples obtained in each synthetic step were thoroughly characterized. The coloration of NH\u003csub\u003e2\u003c/sub\u003e − CNFs support indicates the formation of Ag NPs, and the optical changes induced by the appearance of Ag NPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Opposite to NH\u003csub\u003e2\u003c/sub\u003e − CNFs support, the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs hybrid absorbs in the visible and near-infrared spectral region with surface plasmon resonance bands peaking around 400 and 550 nm, indicating a relatively small size of metallic silver. The peak at around 400 nm is not well-resolved due to overlap with the absorption of NH\u003csub\u003e2\u003c/sub\u003e − CNFs support.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe TG measurement in the air was applied to evaluate the content of the inorganic phase in the hybrid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The residual mass of CNFs and NH\u003csub\u003e2\u003c/sub\u003e − CNFs samples at high temperatures (\u0026gt; 700 ºC) was negligible. On the other hand, the residual mass of the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs, corresponding to the content of metallic silver, was around 20 wt.-%. Based on the concentration of precursors, free amino groups in NH\u003csub\u003e2\u003c/sub\u003e − CNFs, and Ag\u003csup\u003e+\u003c/sup\u003e ions, the calculated maximal content of metallic silver is 35 wt.-%. The experimentally measured lower inorganic phase content is the most likely consequence of amino groups' steric hindrance during the Ag NPs formation.\u003c/p\u003e \u003cp\u003eThe wide-angle XRD pattern of the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs hybrid, presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, gave diffraction peaks at 38.1, 43.7, 64.3, and 77.3º belonging to the (111), (200), (220), and (311) crystal planes of face-centered-cubic silver (Card No: 9013050). The average crystallite size of silver was estimated to be around 5 nm based on the half-width of diffraction peaks and using Scherrer’s equation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe XRD spectra of CNFs and NH\u003csub\u003e2\u003c/sub\u003e − CNFs, shown in the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, correlated with the literature data [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], exhibiting the principal peaks around 16.5, 22.5, and 35° which is attributed to the overlapping (1–10) and (110), (200) and (004) planes of monoclinic cellulose, respectively. The empirical equation was applied to calculate the crystallinity index (CI) of CNFs and NH\u003csub\u003e2\u003c/sub\u003e − CNFs using the intensity of the highest XRD peak (I\u003csub\u003e002\u003c/sub\u003e) and the minimum intensity at a position between the (002) and the (101) peaks (I\u003csub\u003eAM\u003c/sub\u003e), which was at about 18.3° [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]:\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{C}\\text{I}= \\frac{{\\text{I}}_{002}-{\\text{I}}_{\\text{A}\\text{M}}}{{\\text{I}}_{002}} \\times 100\\text{\\%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003eThe crystallinity indexes of CNFs and NH\u003csub\u003e2\u003c/sub\u003e − CNFs were found to be around 58.3 and 32.7%, indicating that the functionalization steps (above all, the oxidation phase) slightly influenced the crystal-like arrangement of hydrogen-bonded CNFs and consequently their morphology [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe thorough morphological characterization of the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs composite was performed using TEM. Low-magnification TEM images of Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs composite (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, A and B) indicated the presence of randomly distributed spherical nanometer in size (\u0026lt; 10 nm) Ag NPs, although agglomeration is seldom noticeable. So, there was a good agreement concerning the size estimation of Ag NPs between TEM, XRD, and spectroscopy data. Analysis of the selected area electron diffraction (SAED) pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) revealed the presence of diffraction rings consistent with the inverse face-centered-cubic crystalline silver structure, and the analysis of the EDX spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed the presence of a pronounced peak corresponding to silver. Finally, a high-resolution TEM image displays typical Ag NPs attached to NH\u003csub\u003e2\u003c/sub\u003e − CNFs support (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The morphological changes occurring in each step of the amino-functionalization process of CNFs, including the influence of dispersion media, were in detail described in our recent publications [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and are omitted in the present study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well-known that the surface charge of Gram-positive and Gram-negative bacteria is negative in media of different acidities and ionic strengths [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Representative of Gram-negative bacteria, \u003cem\u003eE. coli\u003c/em\u003e, has a more negatively charged cell wall than typical Gram-positive bacteria, \u003cem\u003eS. aureus\u003c/em\u003e. So, it is crucial to determine the pH-dependent surface charge and ζ-potential of all samples (CNFs, NH\u003csub\u003e2\u003c/sub\u003e − CNFs, and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs) from the electrostatic interaction point of view. Also, the potentiometric titration curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and the pH-dependent ζ-potential values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) indicated chemical changes occurring during the synthetic route from CNFs over NH\u003csub\u003e2\u003c/sub\u003e − CNFs to Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs. The native CNFs are negatively charged in the entire pH region, showing a negligible bend of negative charge (0.12 mmol/g) at pH around 4–5, related to the seldom presence of anionic surface groups, preferably carboxylic, formed during the preparation of CNFs or due to residual lignin, responsible for ζ-potential of around − 31.6 mV at pH 7 (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe titration curves (forth and back) of pure HMDA (inset in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) show a steady high positive charge in a broad pH range, up to the pH 10, close to its deprotonation constant (pK = 11; [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]) where a steep decrease takes place. So, the functionalization of CNFs with HMDA, followed by the introduction of amino groups, leads to positively surface-charged NH\u003csub\u003e2\u003c/sub\u003e − CNFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) at pH values lower than 9. Also, the titration curves of the NH\u003csub\u003e2\u003c/sub\u003e − CNFs have different shapes compared to the native HMDA. The gradual increase of positive charge is noticeable by increasing the acidity with a barely noticeable bend in the intermediate pH range, confirming the consumption of one of the amino groups from HMDA during the functionalization of CNFs and yielding a total charge of around 5.99 mmol/g. In particular, at pH 7, where the material property is the most important from the applicative side, the ζ-potentials of the NH\u003csub\u003e2\u003c/sub\u003e − CNFs is + 31.3 mV with 2.44 mmol/g of functional groups. These results agree with our recently published data [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe formation of Ag NPs and their conjugation to functionalized CNFs over amino groups follows a significant decrease in surface charge and ζ-potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, respectively). Consequently, the total charge is about 4.64 mmol/g, and at pH 7, ζ-potential is + 19.9 mV with corresponding 1.0 mmol/g functional groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The presence of remaining free amino groups in Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs indicates non-stoichiometrical conjugation of Ag NP to NH\u003csub\u003e2\u003c/sub\u003e-CNF, and it can be related to the formation mechanism of metallic silver rather than individual conjugation. The pH-dependent potentiometric titration and ζ-potential measurements are in agreement with the thermogravimetric estimation of the content of silver in Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs, found to be lower than expected for the stoichiometric reaction between Ag\u003csup\u003e+\u003c/sup\u003e ions and amino groups.\u003c/p\u003e \u003cp\u003eIn addition, the ζ-potentials and the average size of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs were thoroughly analyzed in mixed solvents, water-ethanol or water-acetone of different compositions, at pH 7, and compared with results obtained in water (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). It was confirmed that the presence of hydrophobic amino-bearing molecules (HMDA contains ethyl units) attached to CNFs induced their aggregation in water, which can be reduced in the presence of highly polar solvents [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. First, the hydrodynamic size analysis of the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs dispersed in water revealed the presence of significantly larger micro-sized particulates (27.7 µm) compared to the NH\u003csub\u003e2\u003c/sub\u003e − CNFs with high polydispersity and average size of 10.5 µm. Generally, the formation of several micrometers in size NH\u003csub\u003e2\u003c/sub\u003e − CNFs is related to a both-sides (crosslinking) attachment of HMDA to pre-oxidized CNFs that also occurred, besides one-side (grafting) reaction, as well as the formation of aggregates due presence of hydrophobic methylene chain in attached HMDA, as already established in our previous studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. On the other hand, a diminished ζ-potential upon the attachment of Ag NPs to the NH\u003csub\u003e2\u003c/sub\u003e − CNFs support, from + 31.3 to + 19.9 mV (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), i.e., the decrease of surface charge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) is responsible for the formation of significantly larger Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs hybrids. However, the differences in the ζ-potentials between NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs are almost negligible for any composition of studied mixed solvents. For example, ζ-potentials of 4.7–3.5 µm large NH\u003csub\u003e2\u003c/sub\u003e − CNFs in miliQ water containing 12, 25, and 50 vol.-% of ethanol are about + 20.0, + 27.0, and + 31.5 mV, respectively, while corresponding values for the 5.9–5.3 µm large Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs are + 23.8, + 28.1, and + 28.5 mV, respectively. As a consequence of the similar ζ-potentials in mixed solvents, the hydrodynamic sizes of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs, determined by the DLS, are close to each other, within experimental errors, significantly smaller compared to being dispersed in water, and in all cases around 5 µm. Opposite to the mixed water-ethanol solvent, the increase of volume percent of aprotic acetone (does not contain O − H group available for H bonding) in water leads to a decrease of ζ-potentials from around + 36 to + 19 mV of both NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs. The hydrodynamic size of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs is less than 5 µm, except for the mixture with the highest content of acetone (50 vol.-%), where both samples have the lowest ζ-potentials (around + 20 mV).\u003c/p\u003e \u003cp\u003eTo evaluate the antimicrobial ability of prepared Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs, minimum inhibition concentrations (MIC) and minimum bactericidal concentrations (MBC) were measured against Gram-positive bacteria \u003cem\u003eS. aureus\u003c/em\u003e, Gram-negative bacteria \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, and fungi \u003cem\u003eC. albicans\u003c/em\u003e, and compared with the antimicrobial ability of NH\u003csub\u003e2\u003c/sub\u003e − CNFs. This dilution method provides concentration-dependent antimicrobial activity measurements of prepared samples diluted two times up to 128 times. The concentrations of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs stock solutions were 15.5 and 24.0 mg/mL, respectively, and the content of silver in the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs was around 20 wt.-%.\u003c/p\u003e \u003cp\u003eThe CNFs have no inherent antimicrobial activity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], so the control sample did not show any antimicrobial activity. As expected, the NH\u003csub\u003e2\u003c/sub\u003e − CNFs display growth inhibition of all studied microbial species. The MIC values obtained in this study with published MIC values in our previous studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Besides the hydrophobic tail, the presence of the protonated, positively charged amino groups is essential for the efficient antimicrobial activity of NH\u003csub\u003e2\u003c/sub\u003e − CNFs since both the hydrophobic and electrostatic interaction with the hydrophobic and negatively charged cell walls leads to increased contact with microorganisms [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], compromising its integrity and leading to leakage of cytoplasmic content and, ultimately, cell lysis, thereby resulting in a bactericidal effect [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The differences in MIC values of NH\u003csub\u003e2\u003c/sub\u003e − CNFs, particularly for Gram-positive bacteria \u003cem\u003eS. aureus\u003c/em\u003e and Gram-negative bacteria \u003cem\u003eE. coli\u003c/em\u003e, are the most likely consequence of the different testing methodologies causing differences in the dispersibility of samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimum inhibition concentrations (MIC) and minimum bactericidal concentrations (MBC) of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003e − CNFs\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eAg/NH\u003csub\u003e2\u003c/sub\u003e − CNFs\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003cp\u003e(mg/mL)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC (mg/mL)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMIC (mg/mL)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMBC (mg/mL)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. aureos\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e~ 3\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. aeruginosa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. albicans\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.24\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emin after 24 h\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e*\u003c/sup\u003eData from reference 32.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e**\u003c/sup\u003eData from reference 31.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003end ‒ not determined\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eBesides MIC, the MBC values for NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs are also collected in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and based on these data, we can conclude the following. First, both composite display growth inhibition of all studied microbial species. On the other hand, the microorganism-killing activity that prevents their colonization occurs either at higher silver concentrations than inhibition or does not take place in the case of \u003cem\u003eS. aureus\u003c/em\u003e in the studied concentration range. Second, the MIC values are higher than the ones obtained for free-standing similar in-size Ag NPs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] or immobilized Ag NPs on glass support [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, MIC and MBC values depend on the morphology (size and shape), preparation method, and surface properties of Ag NPs. So, a similar MIC value against \u003cem\u003eS. aureus\u003c/em\u003e (0.625 mg/mL) is reported by Parvekar \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] for 5 nm in size Ag NPs, while Ayala-Núñez \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] demonstrated that MIC and MBC of 10 nm Ag NPs are in concentrations of 1.35 mg/mL against \u003cem\u003eS. aureus\u003c/em\u003e. Third, the MIC values obtained for Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs are lower than those for NH\u003csub\u003e2\u003c/sub\u003e − CNFs. Although free amino groups are present in the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs, most likely due to their steric hindrance, the mechanism of the antimicrobial action of the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs is different, switching to the antimicrobial activity of Ag NPs.\u003c/p\u003e \u003cp\u003eThe antimicrobial action of immobilized Ag NPs can be due to direct contact of microbial species with Ag NPs, immobilized or detached in solution, and with released Ag\u003csup\u003e+\u003c/sup\u003e ions in solution [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The advantage of immobilized over colloidal Ag NPs is, as shown by Lv \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], their higher stability and consequently suppressed oxidation that provides long-term antimicrobial activity. From the practical point of view, small and controlled release of Ag NPs and Ag\u003csup\u003e+\u003c/sup\u003e ions in wastewater is the essential prerequisite for its disinfection. So, we aged the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs composite in water and, after one month, applied the ICP-AES technique to determine the concentration of silver in the supernatant. The ICP-AES measurement revealed that the total concentration of released silver is less than 1% of the content of Ag NPs in Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs composite. The ICP-AES technique does not distinguish the valence state of silver, and since the supernatant was yellowish, the presence of detached Ag NP from the composite in the supernatant was proven by spectroscopy measurements. The absorption spectrum of the supernatant (Supporting Information, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) has the surface plasmon resonance band peaking at 410 nm, characteristic of nanometer-in-size Ag particles. Nevertheless, although the Ag/NH\u003csub\u003e2\u003c/sub\u003e-CNF composite serves as a reservoir of silver, the antimicrobial activity will be present even after the complete release of silver since the NH\u003csub\u003e2\u003c/sub\u003e − CNFs can capture and deactivate the bacteria from wastewater [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well-known that released silver in any form (ionic or metallic) retains its cytotoxicity and ecotoxicity even at a concentration as low as 1.0 mg/L [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. So, a trade-off between release rate and efficiency of the antimicrobial action is a prerequisite for the Ag NPs application as a disinfection agent. To conclude, considering the high content of Ag NPs in the Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs (20 wt.-%), slow release of Ag\u003csup\u003e+\u003c/sup\u003e ions to surrounding media, and negligible detachment of Ag NPs, it seems that the prepared sample is suitable for long-term antimicrobial action and worth further investigating its potential application.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe free amino groups in NH\u003csub\u003e2\u003c/sub\u003e − CNFs provided a simple way to prepare inorganic-organic hybrid particles with a high content of Ag NPs (20 wt.-%). Based on the characterization and antimicrobial activities of prepared samples, several conclusions can be drawn.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cul\u003e \u003cli\u003e \u003cp\u003eIncorporating a nanometer large Ag NPs does not significantly affect the hydrodynamic size, ζ-potential, and surface charge of NH\u003csub\u003e2\u003c/sub\u003e − CNFs, i.e., hybrid retained desirable properties of its organic component.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe inorganic-organic (Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs) hybrid and NH\u003csub\u003e2\u003c/sub\u003e − CNFs display similar antimicrobial activity, although the mechanism of their antimicrobial action is different, i.e., electrostatic interactions via amino groups \u003cem\u003eversus\u003c/em\u003e Ag\u003csup\u003e+\u003c/sup\u003e ions released from Ag NPs.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSlow release of Ag ions and negligible detachment of Ag NPs to surrounding media, i.e., stability of prepared hybrid, ensure its long-term antimicrobial action.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe believe that the obtained results are a good starting point to extend further investigations towards potential applications of hybrid consisting of NH\u003csub\u003e2\u003c/sub\u003e − CNFs and Ag/NH\u003csub\u003e2\u003c/sub\u003e − CNFs for wastewater treatment on a large scale.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has received funding from the Slovenian Research and Innovation Agency (Research Programme P2-0424, Research projects BI-RS 18/19-043). Also, this research was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract Number: 451-03-66/2024-03/200017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowl\u003c/strong\u003e\u003cstrong\u003eedgmen\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Vera Vivod, MSc., for preparing the samples and performing the zeta-size analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare that all the data generated or analyzed during this study are available within the article.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eVesna Lazić:\u0026nbsp;\u003c/strong\u003eInvestigation, Data curation, Methodology, Funding acquisition. \u003cstrong\u003eJovan M. Nedeljković:\u0026nbsp;\u003c/strong\u003eConceptualisation, Methodology, Investigation, Visualisation, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing. \u003cstrong\u003eVanja Kokol:\u0026nbsp;\u003c/strong\u003eConceptualisation, Funding acquisition, Methodology, Investigation, Visualisation, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll of the authors consent to participate in this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll of the authors consent to publish this manuscript\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResearch involving human participants or animals\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eD. Trache, A.F. Tarchoun, M. Derradji, T.S. Hamidon, N. Masruchin, N. Brosse, M.H. Hussin, Nanocellulose: From Fundamentals to Advanced Applications, Front. Chem., 8, 392 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Thomas, M.C. Raj, J. Joy, A. Moores, G.L. Drisko, C. Sanchez, Nanocellulose, a versatile green platform: From biosources to materials and their applications, Chem. Rev., 118, 11575\u0026ndash;11625 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Thomas, T. Duolikun, N.P. Rumjit, S. Moosavi, C.W. Lai, M.R.B. Johan, L.B. Fen, Comprehensive review on nanocellulose: Recent developments, challenges and future prospects, J. Mech. Behav. Biomed., 110, 103884 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Dalei, S. Das, M. Pradhan, Dialdehyde cellulose as a niche material for versatile applications: An overview, Cellulose, 29, 5429\u0026ndash;5461 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Thakur, A. Guleria, S. Kumar, S. Sharma, K. Singh, Recent advances in nanocellulose processing, functionalization and applications: A review, Mater. Adv., 2, 1872\u0026ndash;1895 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Ilić, Z. Šaponjić, V. Vodnik, B. Potkonjak, P. Jovančić, J. Nedeljković, M. Radetić, The influence of silver content on antimicrobial activity and color of cotton fabrics functionalized with Ag nanoparticles, Carbohyd. Polym., 78, 564\u0026ndash;569 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Jia, Y. Mei, L. Cheng, J. Zhou, L. Zhang, Preparation of copper nanoparticles coated cellulose films with antibacterial properties through one-step reduction, ACS Appl. Mater. Inter., 4, 2897\u0026ndash;2902 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY. Xu, S. Li, X. Yue, W. Lu, Review of silver nanoparticles (AgNPs)-cellulose antibacterial composites, BioRes., 13, 2150\u0026ndash;2170 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Mihailović, Z. Šaponjić, M. Radoičić, S. Lazović, C.J. Baily, P. Jovančić, J. Nedeljković, M. Radetić, Functionalization of cotton fabrics with corona/air RF plasma and colloidal TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles, Cellulose, 18, 811\u0026ndash;825 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB.L. Carpenter, F. Scholle, H. Sadeghifar, A.J. Francis, J. Boltersdorf, W.W. Weare, D.S. Argyropoulos, P.A. Maggard, R.A. Ghiladi, Synthesis, characterization, and antimicrobial efficacy of photomicrobicidal cellulose paper, Biomacromolecules, 16, 2482\u0026ndash;2492 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Yoosefi Booshehri, R. Wang, R. Xu, Simple method of deposition of CuO nanoparticles on a cellulose paper and its antibacterial activity, Chem. Eng. J., 262, 999\u0026ndash;1008 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Lefatshe, C.M. Muiva, L.P. Kebaabetswe, Extraction of nanocellulose and in-situ casting of ZnO/cellulose nanocomposite with enhanced photocatalytic and antibacterial activity, Carbohyd. Polym., 164, 301\u0026ndash;308 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.-W. Zhao, C.-R. Guo, Y.-Z. Hu, Y.-R. Guo, Q.-J. Pan, The preparation and antibacterial activity of cellulose/ZnO composite: a review, Open Chem., 16, 9\u0026ndash;20 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT.C. Mokhena, M.J. John, Cellulose nanomaterials: New generation materials for solving global issues, Cellulose, 27, 1149\u0026ndash;1194 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026uuml;lsu, E. Y\u0026uuml;ksektepe, Preparation of gentamicin conjugated cellulose nanocrystals and evaluation of efficacy on different microorganisms, Eur. J. Sci. Tech., 27, 1105\u0026ndash;1112 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Amara, A. El Mahdi, R. Medimagh, K. Khwaldia, Nanocellulose-based composites for packaging applications. Curr. Opin. Green Sust., 31, 100512 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Zhang, P. Yan, Y. Li, X. He, Y. Dai, Z. Tan, Preparation and antibacterial activity of a cellulose-based schiff base derived from dialdehyde cellulose and L-lysine, Ind. Crop Prod., 145, 112126 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. He, Y. Li, L. Zhang, R. Du, Y. Dai, Z. Tan, Preparation of 2,3-dialdehyde microcrystalline cellulose particles crosslinked with ε-poly-L-lysine and their antibacterial activity, Cellulose, 28, 2833\u0026ndash;2847 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Dalei, S. Das, M. Pradhan, Dialdehyde cellulose as a niche material for versatile applications: An overview, Cellulose, 29, 5429\u0026ndash;5461 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Timofeeva, N. Kleshcheva, Antimicrobial polymers: Mechanism of action, factors of activity, and applications, Appl. Microbiol. Biot., 89, 475\u0026ndash;492 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO.M. Bondarenko, M. Heinlaan, M. Sihtm\u0026auml;e, A. Ivask, I. Kurvet, E. Joonas, A. Jemec, M. Mannerstr\u0026ouml;m, T. Heinonen, R. Rekulapelly, S. Singh, J. Zou, I. Pyykk\u0026ouml;, D. Drobne, A. Kahru, Multilaboratory evaluation of 15 bioassays for (eco) toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID, Nanotoxicology, 10, 1229\u0026ndash;1242 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Tavakolian, S.M. Jafari, T.G. van de Ven, A review on surface-functionalized cellulosic nanostructures as biocompatible antibacterial materials, Nano-Micro Lett., 12, 1\u0026ndash;23 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Chen, Y.G. Feng, X. Wang, T.C. Li, J.Y. Zhang, D.J. Qian, Silver nanoparticles capped by oleylamine: formation, growth, and self-organization, Langmuir, 23, 5296\u0026ndash;5304 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Mourdikoudis, L.M. Liz-Marzan, Oleylamine in Nanoparticle Synthesis, Chem. Mater., 25, 1465\u0026ndash;1476 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI.D. Vukoje, E.S. Džunuzović, V.V. Vodnik, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Synthesis, characterization, and antimicrobial activity of poly(GMA-co-EGDMA) polymer decorated with silver nanoparticles, J. Mater. Sci., 49, 6838\u0026ndash;6844 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI.D. Vukoje, E.S. Džunuzović, D.R. Lončarević, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Synthesis, characterization, and antimicrobial activity of silver nanoparticles on poly(GMA-co-EGDMA) polymer support, Polym. Composite, 38, 1206\u0026ndash;1214 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI.D. Vukoje, E.S. Džunuzović, S. Dimitrijević, S.P. Ahrenkiel, J.M. Nedeljković, Size-Dependent Antibacterial Properties of Ag Nanoparticles Supported by Amino-Functionalized Poly(GMA-co-EGDMA) Polymer, Polym. Composite, 40, 2901\u0026ndash;2907 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Lazić, I. Smičiklas, J. Marković, D. Lončarević, J. Dostanić, S.P. Ahrenkiel, J.M. Nedeljković, Antibacterial ability of supported silver nanoparticles by functionalized hydroxyapatite with 5-aminosalicylic acid, Vacuum, 148, 62\u0026ndash;68 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Lazić, K. Mihajlovski, A. Mraković, E. Ill\u0026eacute;s, M. Stoiljković, S.P. Ahrenkiel, J.M. Nedeljković, Antimicrobial activity of silver nanoparticles supported by magnetite, ChemistrySelect, 4, 4018\u0026ndash;4024 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Lazić, V. Vivod, Z. Peršin, M. Stoiljković, I.S. Ratnayake, P.S. Ahrenkiel, J.M. Nedeljković, V. Kokol, Dextran-coated silver nanoparticles for improved barrier and controlled antimicrobial properties of nanocellulose films used in food packaging, Food Packaging Shelf, 26, 100575 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Kokol, V. Vivod, Z. Peršin Fratnik, M. Čolić, M. Kolar, Antimicrobial properties of viscose yarns ring-spun with integrated amino-functionalized nanocellulose, Cellulose, 28, 6545\u0026ndash;6565 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Kokol, S. Novak, V. Kononenko, M. Kos, V. Vivod, N. Gunde-Cimerman, D. Drobne, Antibacterial and degradation properties of dialdehyded and aminohexamethylated nanocelluloses, Carbohyd. Polym., 311, 120603\u0026ndash;120619 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQ. Xu, X. Huang, L. Guo, Y. Wang, L. Jin, Enhancing Removal of Cr(VI), Pb\u003csup\u003e2+\u003c/sup\u003e, and Cu\u003csup\u003e2+\u003c/sup\u003e from Aqueous Solutions Using Amino-Functionalized Cellulose Nanocrystal, Molecules, 26, 7315 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.L. Tummino, E. Laurenti, P. Bracco, C. Cecone, V. La Parola, C. Vineis. M.L. Testa, Antibacterial properties of functionalized cellulose extracted from deproteinized soybean hulls, Cellulose, 30, 7805\u0026ndash;7824 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Park, J.O. Baker, M.E Himmel, P.A. Parilla, D.K. Johnson, Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance, Biotechnol. Biofuels, 3:10 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Errokh, A. Magnin, J.-L. Putaux, S. Boufi, Morphology of the nanocellulose produced by periodate oxidation and reductive treatment of cellulose fibers, Cellulose, 25, 3899\u0026ndash;3911 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Sonohara, N. Muramatsu, H. Ohshima, T. Kondo, Difference in surface properties between \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e as revealed by electrophoretic mobility measurements, Biophys. Chem., 55, 273\u0026ndash;277 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB.K. Mondal, S.S. Bandyopadhyay, A.N. Samanta, Kinetics of CO\u003csub\u003e2\u003c/sub\u003e absorption in aqueous hexamethylenediamine, Int. J. Greenhouse Gas Control, 56, 116\u0026ndash;125 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Rashki, N. Shakour, Z. Yousefi, M. Rezaei, M. Homayoonfal, E. Khabazian, F. Atyabi, F. Aslanbeigi, R.S. Lapavandani, S. Mazaheri, M.R. Hamblin, H. Mirzaei, Cellulose-Based Nanofibril Composite Materials as a New Approach to Fight Bacterial Infections, Front. Bioeng. Biotech., 9, 732461 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Rajagopal, S. Walker, Envelope structures of gram-positive bacteria, Curr. Top. Microbiol. Immunol., 404, 1\u0026ndash;44. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Pan\u0026aacute;ček, L. Kv\u0026iacute;tek, R. Prucek, M. Kol\u0026aacute;ř, R. Večeřov\u0026aacute;, N. Piz\u0026uacute;rov\u0026aacute;, V.K. Sharma, T. Nevěčn\u0026aacute;, R. Zbořil, Silver Colloid Nanoparticles: Synthesis, Characterization, and Their Antibacterial Activity, J. Phys. Chem. B, 110,16248\u0026ndash;16253 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Agnihotri, S. Mukherji, S. Mukherji, Size-controlled silver nanoparticles synthesized over the range 5\u0026ndash;100 nm using the same protocol and their antibacterial Efficacy, RSC Adv., 4, 3974\u0026ndash;3983 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Parvekar, J. Palaskar, S. Metgud, R. Maria, S. Dutta, The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, Biomater. Investig. Dent., 7, 105\u0026ndash;109 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN.V. Ayala-N\u0026uacute;\u0026ntilde;ez, H.H. Lara Villegas, L. del Carmen Ixtepan Turrent, C. Rodr\u0026iacute;guez Padilla, Silver Nanoparticles Toxicity and Bactericidal Effect Against Methicillin-Resistant Staphylococcus aureus: Nanoscale Does Matter, NanoBiotechnology, 5, 2\u0026ndash;9 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Agnihotri, S. Mukherji, S. Mukherji, Immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver, Nanoscale, 5, 7328\u0026ndash;7340 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Lv, S. Su, Y. He, Q. Huang, W. Hu, D. Li, C. Fan, S.-T. Lee, Long-Term Antimicrobial Effect of Silicon Nanowires Decorated with Silver Nanoparticles, Adv. Mater., 22, 5463\u0026ndash;5467 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Kokol, M. Kos, V. Vivod, N. Gunde-Cimerman, Cationised Fibre-Based Cellulose Multi-Layer Membranes for Sterile and High-Flow Bacteria Retention and Inactivation, Membranes,13, 284 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Pan\u0026aacute;ček, M. Kol\u0026aacute;ř, R. Večeřov\u0026aacute;, R. Prucek, J. Soukupov\u0026aacute;, V. Kryštof, P. Hamal, R. Zbořil, L. Kv\u0026iacute;tek, Antifungal activity of silver nanoparticles against Candida spp., Biomaterials, 30, 6333\u0026ndash;6340 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amino-modified cellulose nanofibrils, Silver nanoparticles, Hybrid microparticles, Zeta-potential, Antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-4507463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4507463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe silver nanoparticles (Ag NPs) conjugated with amino-functionalized cellulose nanofibrils (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs) were \u003cem\u003ein situ\u003c/em\u003e prepared by reducing silver ions with free amino groups from NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs. The spectroscopy and transmission electron microscopy measurements confirmed the presence of non-agglomerated nanometer-in-size Ag NPs within micrometer-large NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs of high (20 wt.-%) content. Although the consumption of amino groups during the formation of Ag NPs lowers the ζ-potential and surface charge of prepared inorganic-organic hybrids (from +\u0026thinsp;31.3 to +\u0026thinsp;19.9 mV and from 2.4 to 1.0 mmol/g at pH 7, respectively), their values are sufficiently positive to ensure electrostatic interaction with negatively charged cell walls of pathogens in acidic and slightly (up to pH\u0026thinsp;~\u0026thinsp;8.5) alkaline solutions. The antimicrobial activity of hybrid microparticles against various pathogens (\u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e) is comparable with pristine NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CNFs. However, a long-timescale use of hybrids ensures the slow and controlled release of Ag\u003csup\u003e+\u003c/sup\u003e ions to surrounding media (less than 1 wt.-% for one month).\u003c/p\u003e","manuscriptTitle":"Antimicrobial activity of amino-modified cellulose nanofibrils decorated with silver nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 09:37:06","doi":"10.21203/rs.3.rs-4507463/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df622786-4a29-4b84-9cc1-a023d24a5520","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-22T00:36:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-27 09:37:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4507463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4507463","identity":"rs-4507463","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}