Unraveling the reaction mechanism of silver ions reduction by chitosan from so far neglected spectroscopic features

Carbohydrate Polymers 174 (2017) 601–609

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Unraveling the reaction mechanism of silver ions reduction by chitosan from so far neglected spectroscopic features Ana Patrícia Carapeto ∗ , Ana Maria Ferraria ∗ , Ana Maria Botelho do Rego Centro de Química-Física Molecular and IN, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049 001 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 29 March 2017 Received in revised form 8 June 2017 Accepted 26 June 2017 Available online 28 June 2017 Keywords: Chitosan Nanoparticles Silver UV–vis XPS AFM

a b s t r a c t Metallic silver nanoparticles were synthesized in aqueous solution using chitosan, as both reducing and stabilizing agent, and AgNO3 as silver precursor aiming the production of solid ultra-thin films. A systematic characterization of the resulting system as a function of the initial concentrations was performed. The combination of UV–vis absorption – and its quantitative analysis – with X-ray photoelectron spectra, light scattering measurements and atomic force microscopy allowed obtaining a rational picture of silver reduction mechanism through the identification of the nature of the formed reduced/oxidized species. Nanoparticle mean sizes and sizes distributions were rather independent from the precursors initial absolute and relative concentrations ([AgNO3 ]/[chitosan]). This work clarifies some points of the mechanism involved showing experimental evidence of the early stages of the very fast silver reduction in chitosan aqueous solutions through the spectral signature of the smallest silver aggregate (Ag2 + ) even at room temperature. The characterized system is believed to be useful for research fields where silver nanoparticles completely exempt of harmful traces of inorganic ions, coming from additional reducing agents, are needed, especially to be used in biocompatible in films. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Hybrid systems combining chitosan with metal nanoparticles, like silver nanoparticles (Ag NP), have been studied for over a decade, finding applications, in catalysts (Calo et al., 2004; Rajesh, Sujanthi, Kumar, & Venkatesan, 2015), chemiluminescent sensors and biosensors (Wang & Cui, 2008; Zhang et al., 2011), optical sensors or waveguides (Mironenko, Modin, Sergeev, Voznesenskiy, & Bratskaya, 2014), bactericide nanocomposites, or phototherapeutic agents (Sanpui, Murugadoss, Prasad, Ghosh, & Chattopadhyay, 2008; Boca et al., 2011), just to mention a few. Chitosan is a biocompatible and biodegradable, naturally occurring polysaccharide produced by deacetylation of chitin. Scheme 1 shows its chemical structure, with some degree of acetylation (chitin-like). Chitosan is known to be soluble in (slightly acidic) aqueous solutions, being also a chelating agent that can be used as both a sta-

∗ Corresponding authors at: CQFM and IN, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. E-mail addresses: [email protected] (A.P. Carapeto), [email protected] (A.M. Ferraria), [email protected] (A.M.B. do Rego). http://dx.doi.org/10.1016/j.carbpol.2017.06.100 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

bilizing and a reducing agent of metal ions (Pestov, Nazirov, Modin, Mironenko, & Bratskaya, 2015; Huang & Yang, 2004). On the other hand, silver nanoparticles have, among other advantages, unique antimicrobial properties that have led to their success in biomedical applications (Rai, Yadav, & Gade, 2009). However, metal NP are prone to aggregation unless stabilized. Recently, in our group, silver and gold metallic NP were synthesized in situ on aminated cellulose ultrathin films and fibers (Ferraria, Boufi, Battaglini, Botelho do Rego, & Vilar, 2010; Boufi et al., 2011). It was shown that Ag and Au NP nucleate and grow directly at the surface of cellulose previously functionalized with amine groups. Most importantly, the surface was efficiently modified after interaction with metal salts without adding any external reducing agent and the reaction took place selectively and exclusively at the surface, leaving the liquid phase exempt from NP. The most common method used to synthesize Ag NP in the presence of chitosan is the chemical reduction of the metal salt using reducing agents such as NaBH4 , citrate, ascorbic acid or a combination of different reagents (Huang, Yuan, & Yang, 2004; Potara, Gabudean, & Astilean, 2011). However, some of the reagents used may have several harmful effects on the environment and human health. Alternatively, several processes involving non-toxic reducing agents have been proposed. For instance, the use of glucose followed by a microwave thermal treatment (Bozanic,

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Scheme 1. Chemical structure of chitosan with a fraction y/(x + y) of acetylated moieties.

Trandafilovic, Luyt, & Djokovic, 2010) takes advantage of the presence of the hemiacetal (cyclic form)/aldehyde (open form) moiety. In another approach, Rodríguez-Argüelles et al. were able to synthesize tiny Ag NP (<1.7 nm) on a chitosan-tripolyphosphate (TPP) nanocomposite, after a thermal treatment (Rodríguez-Argüelles, Sieiro, Cao, & Nasi, 2011). Photon-assisted reduction methods have also been reported, in particular by ␥-rays (Chen, Song, Liu, & Fang, 2007) or UV-irradiation (Reicha, Sarhan, Abdel-Hamid, & ElSherbiny, 2012). In most of these studies, the efficiency of the Ag0 nanoparticles synthesis is attested by its typical surface plasmon resonance absorbance close to 400 nm, XRD patterns and TEM images. For instance, Wei et al. synthesized chitosan-stabilized Ag NP by heat treatments and using only chitosan as reducing and stabilizing agent, a green chemistry approach identical to the one described here. Systems were studied mainly by FTIRS, TEM and UV–vis absorption spectroscopy (Wei, & Qian, 2008). However, no quantitative data treatment is performed. Actually, in most publications, in spite of hypothesizing about the probable intermediate species involved in the silver reduction mechanism, just a few show experimental evidence supporting the existence of those species at some point of the reaction or under given conditions. The exception is the Reicha and collaborators’ study that shows spectral features assigned to Ag+ -Chitosan complexes formed by an electrochemical process before the UV irradiation. Here, Ag NP were generated, in situ, from the interaction between a silver salt and the chitosan surface, under a mild thermal treatment, and in the absence of any additional reducing agent, stabilizer, template or activating irradiation. The hypothesis here stated is that a suitable combination of the quantitative UV/Vis absorption spectra with X-ray Photoelectron Spectra (XPS) data treatments will clarify the correlation (if any) between the NP yield and the chitosan and silver nitrate concentrations (both absolute and relative). The present work aims: to unravel the mechanism of silver reduction detecting the chemical species produced at the early stages of the interaction between the silver salt and chitosan, with the help of a detailed data treatment of both UV/Vis absorption and XPS; to identify the final oxidation states of silver by an appropriate XPS data treatment (none of the studies reported on these hybrid systems ever made a suitable oxidation state identification/quantification, even those where XPS analysis was used) and, in this way, to evaluate the reduction extent of Ag+ to Ag0 . System characterization also shed some light on the chitosan oxidation mechanism. The characterization was complemented by DLS and AFM measurements. 2. Experimental 2.1. Materials Chitosan low molecular weight with Mw 50,000–190,000 Da and a degree of deacetylation ≥75% (according to the Aldrich specification sheet), estimated by XPS to be ∼79%, was used. For the

work here reported, the chitosan used for all experiments came from exactly the same pack, therefore the Mw and the degree of deacetylation is kept constant. Silver nitrate 99.9999% trace metals basis and acetic acid ACS reagent ≥99.7% were purchased from Aldrich and used without further purification. Acetone, UVASOL for spectroscopy ≥99.8% was obtained from Merck. Solutions were prepared using deionized water (DIW) with a resistivity of 18.2  ·cm, supplied by a MILLIPORE system fed with distilled water. The glass substrates (Corning Glass) were acquired to Aldrich. 2.2. Sample preparation Glass substrates were rinsed with acetone and dried with an argon flow. Aqueous chitosan/AgNO3 solutions of chitosan (1, 3, 5 and 7 mg/mL) in acid medium (1% V/V acetic acid, pH = 5) and 1.0 × 10−2 M in AgNO3 were prepared. Solutions of 7 mg/mL in chitosan and AgNO3 with six further concentrations (4.0 × 10−4 M; 1.0 × 10−3 M, 4.0 × 10−3 M; 2.0 × 10−2 M; 3.0 × 10−2 M or 4.0 × 10−2 M) were also prepared. To obtain Chitosan/Ag nanoparticles solutions, the above-mentioned mixtures were stirred for 48 h at 95 ◦ C, in total darkness. Solutions were prepared as follows: in a volumetric flask of 100 mL, an aqueous acetic acid solution 1% (V/V) was prepared; next, the appropriate mass (20, 60, 100 or 140 mg) of chitosan, in a flask of 20 mL, was mixed with acid solution to attain a volume <10 mL. This set was stirred overnight. Once the chitosan solution was transparent, 10 mL of AgNO3 solution prepared in the acetic acid solution and having a concentration twice the target value was added to the chitosan solution and, finally, the needed acid solution was added to complete 20 mL. All the concentrations referred in the text are the chitosan/AgNO3 concentrations in the final solution (20 mL). Samples studied in the film form were prepared as follows: For XPS characterization, chitosan/NP suspensions were dropped on glass substrates and analyzed after solvent evaporation. Asreceived chitosan powder was also analyzed by XPS for comparison purposes. Chitosan/Ag NP thin films for AFM analyses were spin-coated (speed = 2000 rpm, t = 60 s, acceleration = 800 rpm/s) on glass substrates. A Photo-Resist Spinner (Headway Research Inc.) spin-coating apparatus was used for the deposition of the films. 2.3. Characterization The obtained chitosan/NP solutions were used immediately after 48 h of interaction at 95 ◦ C, for the UV–vis absorption spectroscopy and light scattering characterization. UV–vis absorption spectra were measured at room temperature using a Jasco (Easton, MD) V-560 spectrophotometer and corrected for light-scattering artifacts by subtracting the respective blank sample (acetic acid/water 1% V/V). The hydrodynamic diameter of nanoparticles in solution was determined by Dynamic Light Scattering (DLS) using a ZetaSizer Nano (ZS 90, Malvern, Inc.) analyzer at room temperature. A He-Ne laser (633 nm) was used as a light source and the intensity of scattered light was measured at 90◦ . In these measurements, 12 mm square disposable polystyrene cells (DTS0012, Malvern, Inc.) were used. XPS analyses were performed with a XSAM800 (KRATOS) spectrometer operating in the fixed analyzer transmission (FAT) mode, with a pass energy of 20 eV and a non-monochromatized Mg K␣ radiation (h = 1253.6 eV) produced with a power of 120 W. Data were recorded by the software Vision 2 for Windows, Version 2.2.9 from KRATOS, with a step of 0.1 eV. No flood gun was used for charge correction. The binding energy (BE) was corrected considering the charge shift observed for the sp3 C C and C H peak that should be

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Fig. 1. UV–vis absorption spectra for the aqueous chitosan solution 7 mg/mL (continuous line), for the aqueous [AgNO3 ] = 4 × 10−2 M (long dashed line), for the freshly prepared mixture where [chitosan] = 7 mg/mL and [AgNO3 ] = 4 × 10−2 M (dotted line) and 48 h later (dash-dotted line), at room temperature. For comparison purposes, the sum of the chitosan and AgNO3 spectra is shown (green curve) as well as the difference between the freshly prepared mixture and the Sum (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

centered at 285 eV. Other data treatment details were as described in (Boufi, Rei Vilar, Parra, Ferraria, & Botelho do Rego, 2008). For quantification purposes, sensitivity factors (from Vision 2 library) were 0.318 for C 1s, 0.736 for O 1s, 0.505 for N 1s, and 6.345 for Ag 3d. Morphologic features of spin-coated chitosan/NP films were characterized by a Bruker Innova Atomic Force Microscope (AFM) in tapping mode under ambient conditions with TESP tips, Bruker AFM Probes. The AFM images were analyzed with the software NanoScope Analysis 1.5 from Bruker. 3. Results and discussion 3.1. UV–vis absorption studies Fig. 1 shows the UV–vis absorption spectra for the aqueous chitosan (Chi) solution 7 mg/mL, for the aqueous AgNO3 4 × 10−2 M, and for two mixtures of Chi and AgNO3 , one freshly prepared and another after 48 h at room temperature. For both, [chitosan] = 7 mg/mL and [AgNO3 ] = 4 × 10−2 M. Chitosan solution spectrum displays an increasing absorption from around 590 nm to around 300 nm, reaching a plateau which extends till ∼260 nm and increasing again for lower wavelengths reaching saturation below 250 nm. The AgNO3 aqueous solution absorption spectrum exhibits a peak with a maximum at 304 nm very typical of nitrate ion (Bravo, Olwiert, & Oelckers, 2009). The freshly prepared mixture, containing, simultaneously, chitosan and AgNO3 , shows a spectrum that is not simply the sum of the individual components of the mixture (green curve), showing that the first stage of the reaction is very fast and is responsible for the appearance of one peak at 262 nm and a broad band centered around 334 nm (“Difference” red curve). The peak at 262 nm, as will be explained hereinafter, is due to the oxidation product of chitosan (carbonyl group). The band around 334 nm is, in principle, and accordingly to literature, related with small charged clusters of Ag2 + (Ozin & Hugues, 1983). The same mixture was kept at room temperature in the dark and analyzed 48 h later. The absorbance spectrum is also displayed in Fig. 1 showing that, even at room temperature, the Plasmon absorbance (above 400 nm), due to the silver NP formation, is already present although the absorbance is modest.

Fig. 2(a) shows the raw absorption spectra for aqueous solutions of chitosan having [Chi] = 7 mg/mL and variable [AgNO3 ] after heating at 95 ◦ C, in the dark, for 48 h. Fig. 2(b) shows the same spectra subtracted from the chitosan and the nitrate solution spectra, presented in Fig. 1, assuming that all the nitrate ions remain in solution after the Ag+ reduction and that the Lambert-Beer law applies. This subtraction reveals that the “responsible” for the absorbance increase in this region is a band which peaks at ∼262 nm. The main band appears around 424 nm and is assigned to the Ag NP Surface Plasmon Resonance (SPR) absorption, its position being quite invariable with [AgNO3 ]. Also, the shape of the band, at least for the more concentrated solutions, is rather constant. Fig. 2(c) displays subtracted spectra (as in Fig. 2(b)) for aqueous solutions with constant concentration of AgNO3 (1 × 10−2 M) and variable chitosan concentrations. Once again, the features found in these spectra are the same as in Fig. 2(b). In principle, the invariable SPR position and shape mean that the average and the distribution of nanoparticle sizes are very independent from the [AgNO3 ] and just the absorbance maximum increases revealing that the number of formed NP increases. A recent study on silver NP capped with sodium citrate and tannic acid shows UV–vis absorption spectra with an absorption band similar to the one presented in this work and peaking at 427.5 nm for NP having diameters of 44 nm (Bastús, Piella, & Puntes, 2016). The origin of the band with a maximum at ∼262 nm should correspond to a ␲* ←− n transition in a carbonyl group in an aldehyde or a ketone (Roberts & Caserio, 1967; Williams & Fleming, 1980) that may appear in the medium as an oxidation product of several functional groups in the chitosan. To help the description of all the oxidation half-reactions that can occur in the chitosan, carbon and oxygen atoms numbering is presented in Scheme 2: The reduction and oxidation reactions (involving an oxygen containing group) are described by the following equations: Reduction half-reaction: Ag+ (aq.) + 1 e– → Ag0 Oxidation half-reactions (in each chitosan ring): • Primary alcohol:

(1)

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Fig. 2. UV–vis absorption spectra for (a) the aqueous chitosan solution 7 mg/mL, and variable AgNO3 concentrations; (b) the same solutions after the subtraction of chitosan and NO3 − contributions for spectra (for assumptions, please, see text); (c) subtracted spectra (as in (b)) for aqueous solutions with constant concentration of AgNO3 (1 × 10−2 M) and variable chitosan concentrations; (d) photographs of three of the solutions studied in (c).

C(C3) = O (aq.) + 2 e– + 2 H+ (aq.) ←− HC(C3) –O–H (aq.)

(3)

(ketone)

• Glycosidic bond:

C

(C4’) (ketone∗ )

= O (aq.) + HC(C1) –O–H(aq.) + 2 e– + 2 H + (aq.)

←− HC(C4’) –O–C(C1) H (aq.) + H2 O

*The other hypothesis should be:

Scheme 2. Numbered chitosan atoms.

C(C1) = O (aq.) + HC(C4’) –O–H(aq.) + 2 e– + 2 H + (aq.)

HC(C5) –C(C6) H = O(aq.) + 2 e– + 2 H + (aq.)

(ester)

(aldehyde)

←− HC(C5) –C(C6) H2 –O–H (aq.)

• Secondary alcohol:

(4)

(2)

←− HC(C4’) –O–C(C1) H (aq.) + H2 O

(4 )

Since C1 is bound to O5, the oxidized species should be an ester group rather than a carbonyl one.

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Fig. 3. A424 /A262 represented as a function of the product [AgNO3 ]2 × [Chitosan]. The symbol  represents the values obtained for a constant [Chitosan] and the symbol 䊏 represents the values for a constant [AgNO3 ]. The black line is just to guide the eyes: it has no physical meaning.

Finally, a carbonyl group in an aldehyde may be oxidized to a carboxylic group (Eq. (5)). • Carbonyl (in an aldehyde group) to a carboxyl group: HC(C5) –C(C6) OOH (aq.) + 2 e– + 2 H + (aq.) ←− HC(C5) –C(C6) HO (aq.) + H2 O

(5)

The amino group may also undergo nucleophilic addition (Singh, Narvi, Dutta, & Pandey, 2006) with ketone and/or aldehyde groups formed by oxidation to give hemiaminals which then dehydrate to give substituted imines. Eqs. (1)–(4) show that for each two reduced Ag+ ions, one carbonyl group should be produced. Moreover, Eqs. (2)–(5) show that the chitosan oxidation is favoured in basic medium, fact that was used by Herna´ındez-Mun˜oz group to promote the formation of silver NP in chitosan films prepared with AgNO3 (López-Carballo, Higueras, Gavara, & Herna´ındez-Mun˜oz, 2013).  Since the total number of reduced Ag+ ions is given by

Ni Vi ,

i

Ni being the number of NP of volume Vi , the  number of appearing carbonyl groups should be proportional to

number of NP should be



Ni Vi whereas the

i

Ni .

i

Therefore, A262 (absorbance at 262 nm), assuming that the proformed from Eqs. (2)–(4) keeps constant, portion of carbonyl groups  should be proportional to

Ni Vi whereas A424 , assuming similar

i

size distributions, should be proportional to



Ni .

i

In Fig. 3, A424 /A262 is represented as a function of the product [AgNO3 ]2 × [Chitosan]. Fig. 3 shows that the absorbance ratio increases from ∼0.4 to 1.4. If, for reasoning simplification, a NP monodisperse size distribution existed, then: A424 /A262 ∝ 1/VNP ⇒A424 /A262 ∝ 1/r3NP

605

Consequently, if the change of the ratio A424 /A262 was due to a change of size of the formed NP, the NP radius should change by a factor of (1.4/0.4)1/3 ≈ 1.5 and, moreover, the largest ones should be the ones at low product of concentrations. Given the large steadiness of the SPR band ␭max and shape, this is not a very credible hypothesis. However, in this reasoning, we did not account for the disappearance of carbonyls (in aldehyde groups) by oxidation to carboxyl groups, as described by Eq. (5) and by the reaction with amine groups yielding imine groups. In the carboxyl group, the ␲* level increases but the n (lone pair) level keeps basically unaltered. As a consequence, the energy for the transition ␲* ←− n increases and the corresponding wavelength decreases to around 200 nm falling in a wavelength inaccessible in most spectrophotometers (Williams & Fleming, 1980). In fact, if the carbonyl groups (aldehydes or ketones) resulting from the reaction of AgNO3 with chitosan to form Ag NP kept that form, the proportion of carbonyl groups to Ag NP would be constant, regardless the [AgNO3 ]2 × [Chitosan] product. However, as shown above, at a given point of the reaction, i.e. when the concentration of carbonyl groups is high enough, further reactions are likely to occur: namely the oxidation of aldehydes to carboxyl groups and the nucleophilic addition of aldehydes or ketones with amines to give imines. As a consequence, the proportion between the number of carbonyl groups and the number of formed NP is not constant as shown in Fig. 3. Another aspect (that cannot be explored quantitatively) is that the small aggregates as those detected in the early stages of interaction may still remain after 48 h, when the extent of silver reduction is low. In fact, for those solutions, the band peaking at ∼262 nm is wider for the larger wavelengths side, where, as mentioned above, small aggregates as Ag2 + or even larger, as reported by Bozanic et al. (Bozanic et al., 2010), are detected. The tail resulting from these contributions may increase the absorption maximum at ∼262 nm explaining, at least partly, the behavior found for A424 /A262 for small extents of the silver reduction. 3.2. Dynamic light scattering Fig. 4 shows the results of Dynamic Light Scattering for two different collections of samples prepared with chitosan: the first collection corresponds to aqueous solutions with constant concentration of AgNO3 (1.0 × 10−2 M) and variable chitosan concentrations (1 mg/mL, 3 mg/mL, 5 mg/mL and 7 mg/mL) and the second one to an aqueous 7 mg/mL chitosan solution and variable AgNO3 concentrations (1.0 × 10−2 M, 2.0 × 10−2 M and 3.0 × 10−2 M). The different solutions were analyzed as-prepared: no further salt addition or filtration was performed. The aim was to observe the hybrid objects with the chitosan chains dynamics only affected by the silver nanoparticles (and/or Ag+ ions) chelation (through chitosan amide, amine, alcohol and specially the carbonyl groups formed by oxidation). The measured sizes, crossed with AFM observations (below) and the UV–vis absorption results (above), clearly show that the particles observed are rather chitosan chains capping the silver NP. Since the input parameters in the equipment were not necessarily adequate to the formed hybrid material, absolute values of dynamic diameter in Fig. 4 may be meaningless, but the variation is significant. From UV–vis results, a mean diameter of Ag NP around 44 nm was inferred (Bastús et al., 2016). Assuming a cubic face centered crystalline structure, which has only 74% of occupied space, and spherical NP, the number of reduced silver atoms, NAg0 , in each one should be: NAg 0 =

NPvolume × 0.74 = Atomic volume

4 RNP 3 × 0.74 3 4 3 RMetallic 3 Ag

≈ 2.64 × 106

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Fig. 4. Results of Dynamic Light Scattering: (a) Typical dynamic light scattering distribution curves of the particles hydrodynamic diameter for aqueous solutions with constant concentration of AgNO3 (1 × 10−2 M) and variable chitosan concentrations (left) and aqueous 7 mg/mL chitosan solution and variable AgNO3 concentrations (right); (b) Representation of the particles hydrodynamic diameter (nm) as a function of the nominal [Chitosan]/[AgNO3 ] ratios. Squares and stars correspond to constant [Chitosan] = 0.0484 M and [AgNO3 ] = 0.01 M, respectively.

bonds (Eqs. (4) and (4 )) and, consequently, to the decrease of the average chitosan mean molecular weight. 3.3. X-ray photoelectron spectroscopy

Scheme 3. Chitosan chains surrounding Ag nanoparticles in the samples (a) with low N/Ag; (b) with high N/Ag.

Even for the larger chitosan chain (Mw = 190,000 Da), the maximum number of groups with ability to be oxidized (∼Mw /169.8 × 4 = 4475) is not enough for producing a nanoparticle with the average dimension of 44 nm. Moreover, the total number of chains per NP is a linear function of the atomic ratio N/Ag in the medium. As a consequence, each nanoparticle may be capped (essentially through the groups resulting from the chitosan oxidation) by several chitosan chains, their density at the NP surface being higher for larger N/Ag ratios. In the samples where a smaller number of chains surround each particle (low N/Ag), each chain has space enough to curl around the surface (Scheme 3(a)). Contrarily, for extremely high N/Ag ratio, the NP will be capped by the chitosan chains in a brush-like configuration (Scheme 3(b)). The result will be that the capped NP diameter will increase with the N/Ag ratio. Alternatively, these DLS results are an indication that the average molecular weight of the chitosan chains decreases when a larger number of oxidations occur. This means that one of the intervening reactions is the one leading to the destruction of glycosidic

Aqueous solutions of chitosan (7 mg/mL) and AgNO3 with different concentrations (4.0 × 10−4 M, 4.0 × 10−3 M, 4.0 × 10−2 M) were deposited on glass substrates by simple drop-coating. The coated glass substrates were dried in a vacuum oven at room temperature. The relevant XPS regions of these samples and chitosan (pristine powder) are presented in Fig. 5. All the N 1s XPS regions display a main peak centered at 399.6 ± 0.1 eV assigned to chitosan amine and/or amide groups, and a smaller peak, at higher binding energies (∼402 eV), attributed to protonated amine groups, here abbreviated to “N+ ” (Fig. 5, N 1s). While the atomic ratio N/(chitosan carbons) keeps constant, the relative amount of N+ decreases after interaction with AgNO3 and for increasing concentrations: N+ /NChitosan = 0.17, 0.10 and 0.06 for [AgNO3 ] = 0 M, 4 × 10−4 M and 4 × 10−3 M, respectively, and is negligible for the most concentrated one. In addition, another peak is detected at ∼407 eV. It is assignable to nitrogen atoms from nitrate ions and is particularly intense for the sample prepared from the most concentrated solution in silver nitrate being hardly detected in the sample prepared with the smallest concentration of silver nitrate. The Auger parameters (AP) computed from the Ag 3d photoelectron region and the Ag MNN Auger structure (Fig. 5, Ag 3d and Ag MNN) show that not all the silver is reduced: the sample prepared from the more concentrated solution has an Auger parameter typical of Ag+ (AP-Ag 3d5/2 , Ag Mx N45 N45 = 722.8 eV (for x = 4) or 716.8 eV (for x = 5)), whereas less concentrated solutions seem to have a mixture of Ag0 and Ag+ species (AP-Ag 3d5/2 , Ag Mx N45 N45 = 724.9 eV or 719.0 ± 0.2 eV) (Ferraria, Carapeto, &

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Fig. 5. XPS C 1s, N 1s, Ag 3d and Ag MNN regions of, from bottom to top: Chitosan, Chitosan [7 mg/mL] + AgNO3 [from 4 × 10−4 M to 4 × 10−2 M]. Spectra were offset for clarity sake. AP: Auger Parameters.

Botelho do Rego, 2012). Therefore, even in the sample prepared from the solution with a larger chitosan concentration and the lowest [AgNO3 ], it is not sure that all the silver was reduced. C 1 s regions were fitted with four components typical of those found in pristine chitosan, centered at 285.0 eV, 286.0 eV, 286.7 eV and 288.2 eV (± 0.1) assigned to aliphatic C C and C H, C N, C O and O C O, respectively (Fig. 5, C 1s). The peak at 288.2 eV can include not only carbon C1 in the chitosan ring but also carbon in (N) C O from acetylated moieties or carbon from carbonyl groups. Such carbon atoms can be found between 287.8 and 288.6 eV (Beamson & Briggs, 1992). Quantitatively, if only completely deacetylated chitosan existed (y = 0 in Scheme 1), the

atomic ratio C288.2 /“C-O”286.7 should be 1/4, however, a slight excess of C288.2 was computed for the less concentrated solution ([AgNO3 ] = 4 × 10−4 M) and a substantial excess for the more concentrated one ([AgNO3 ] = 4 × 10−2 M): C288.2 /“C-O”286.7 = 0.27 and 0.45, respectively, indicating that when a large concentration of silver NP exists, also a large increase in carbonyl groups is detected, corroborating the hypotheses made above.

3.4. Atomic force microscopy The procedure to obtain continuous films of chitosan on glass slides by the spin-coating method was optimized and

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Fig. 6. (a) Topographic AFM image with 1 ␮m × 1 ␮m of a chitosan film with silver nanoparticles obtained from a solution with [Chitosan] = 7 mg/mL and [AgNO3 ] = 3 × 10−2 M, (Rq = 4.16 nm); (b) profile of heights (from the white line); (c) histogram of NP height average, mean value = 22 nm.

described on a previous work (Carapeto, Ferraria, & Botelho do Rego, 2015). Since UV–vis absorption results give clear indication that both the size and distribution of silver NP keep constant in all the different conditions here studied, just a single silver concentrated solution ([Chitosan] = 7 mg/mL = 4.8 × 10−2 M and [AgNO3 ] = 3 × 10−2 M) was used to spin-coat a glass substrate and image the respective surface by AFM. Fig. 6 shows the topographic image, the height profile (for a given segment); and the histogram of NP height average. A large density of NP can be observed. The histogram of NP height average was obtained with the software NanoScope Analysis 1.5 from Bruker using the data of the 1 ␮m × 1 ␮m image. The distribution of sizes (with FWHM ∼9 nm) is quite narrow and has a mean value of 22 nm. Since light scattering results are compatible with Ag NP capped with chitosan chains, this last value is likely to give just the height of a half sphere, i.e., the NP radius. This is compatible with the average value inferred from the UV–vis absorption spectra above. 4. Conclusions This green chemistry process does not involve the use of any toxic chemicals, it is cost-effective and environment friendly. Moreover, it does not require the use of further stabilizers, chitosan

playing also that role. In this work, we put in evidence that the products of oxidation in this process are carbonyl groups issued from the oxidation of alcohol and or glycosidic groups, present in the chitosan chains, fully explaining, qualitatively and quantitatively the UV–vis absorption band peaking at ∼262 nm, which is omitted in the few publications dealing with the same system. XPS also confirms a large increase in the amount of carbonyl groups in the medium as the main oxidation product. This fact does not allow electing one of the oxidation mechanisms as being the selective one. DLS results allowed having a rational picture of the silver nanoparticle capping structure by chitosan and, alternatively, they show that oxidation proceeds with chain breaking, showing that at least Eq. (4) occurs. Moreover, XPS shows that the protonated nitrogen (assigned to the chitosan amine since the amide one is a weaker base) almost disappears while the total chitosan nitrogen keeps constant. This is compatible with the amino group undergoing nucleophilic addition with the ketone and/or aldehyde groups formed by oxidation to give imine groups. Although amines are moderately strong bases (pKa ≈ 9), imines are much weaker bases (pKa ≈ 4) (Gennaro, 2006). Finally, XPS showed that, for samples prepared from more concentrated AgNO3 solutions, the most part of the silver “seen” in XPS, corresponding to the most dispersed phase of silver is not Ag0 but rather Ag+ , and, for samples prepared with less concentrated AgNO3 solutions, a mixture of both

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oxidation states exists. This aspect is not usually approached in the literature and opens the way to clarify the old but still current issue about the active species in applications such as bactericide systems. Summarizing, for a given chitosan concentration, even if the ratio N/Ag largely exceeds the stoichiometric one, for increasing silver nitrate concentrations, the yield of the reduction process (NPs formation) decreases, and the fraction of Ag+ final concentration in the medium increases. Silver nanoparticles synthesized by a green process are highly biocompatible and competitive for pharmaceutical and biomedical applications. This process can be easily scaled up for the industrial synthesis of silver nanoparticles namely in the production of bandages since the compatibility of chitosan with the most common biopolymer used in fabrics – cellulose – is complete. Acknowledgements The authors acknowledge funding from Fundac¸ão para a Ciência e a Tecnologia (FCT): A. P. Carapeto Grant SFRH/BD/75734/2011, A. M. Ferraria Grant SFRH/BPD/108338/2015 and project UID/NAN/50024/2013. We also acknowledge North Atlantic Treaty Organization (NATO) SFP project 984842 (CATALTEX) for the AFM (Innova, BRUKER) acquisition. References Bastús, N. G., Piella, J., & Puntes, V. (2016). Quantifying the sensitivity of multipolar (Dipolar, Quadrupolar, and Octapolar) surface plasmon resonances in silver nanoparticles: The effect of size, composition, and surface coating. Langmuir, 32(1), 290–300. Beamson, G., & Briggs, D. (1992). High resolution XPS of organic polymers: The scienta ESCA300 database. Chichester: John Wiley & Sons. Boca, S. C., Potara, M., Gabudean, A. M., Juhem, A., Baldeck, P. L., & Astilean, S. (2011). Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible: Highly effective photothermal transducers for in vitro cancer cell therapy. Cancer Letters, 311, 131–140. Boufi, S., Rei Vilar, M., Parra, V., Ferraria, A. M., & Botelho do Rego, A. M. (2008). Grafting of porphyrins on cellulose nanometric films. Langmuir, 24, 7309–7315. Boufi, S., Ferraria, A. M., Botelho do Rego, A. M., Battaglini, N., Herbst, F., & Vilar, M. R. (2011). Surface functionalisation of cellulose with noble metals nanoparticles through a selective nucleation. Carbohydrate Polymers, 86, 1586–1594. Bozanic, D. K., Trandafilovic, L. V., Luyt, A. S., & Djokovic, V. (2010). ‘Green’ synthesis and optical properties of silver-chitosan complexes and nanocomposites. Reactive and Functional Polymers, 70, 869–873. Bravo, M., Olwiert, A. C., & Oelckers, B. (2009). Nitrate determination in chilean caliche samples by UV-visible absorbance measurements and multivariate. Journal of the Chilean Chemical Society, 1, 93–98. Calo, V., Nacci, A., Monopoli, A., Fornaro, A., Sabbatini, L., Cioffi, N., et al. (2004). Heck reaction catalyzed by nanosized palladium on chitosan in ionic liquids. Organometallics, 23, 5154–5158. Carapeto, A. P., Ferraria, A. M., & Botelho do Rego, A. M. (2015). Chitosan thin films on glass and silicon substrates. Microscopy and Microanalysis, 21(S5), 13–14.

609

Chen, P., Song, L., Liu, Y., & Fang, Y. E. (2007). Synthesis of silver nanoparticles by ␥-ray irradiation in acetic water solution containing chitosan. Radiation Physics and Chemistry, 76, 1165–1168. Ferraria, A. M., Boufi, S., Battaglini, N., Botelho do Rego, A. M., & Vilar, M. R. (2010). Hybrid systems of silver nanoparticles generated on cellulose surfaces. Langmuir, 26, 1996–2001. Ferraria, A. M., Carapeto, A. P., & Botelho do Rego, A. M. (2012). X-ray photoelectron spectroscopy: Silver salts revisited. Vacuum, 86, 1988–1991. Gennaro, A. R. (2006). Organic pharmaceutical chemistry. In D. B. Troy, & P. Beringer (Eds.), Remington: The science and practice of pharmacy (p. 395). Philadelphia: Lippincott Williams & Wilkins. Huang, H., & Yang, X. (2004). Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of tripolyphosphate. Biomacromolecules, 5, 2340–2346. Huang, H., Yuan, Q., & Yang, X. (2004). Preparation and characterization of metal-chitosan nanocomposites. Colloids and Surfaces B: Biointerfaces, 39, 31–37. López-Carballo, G., Higueras, L., Gavara, R., & Herna´ındez-Mun˜oz, P. (2013). Silver ions release from antibacterial chitosan films containing in-situ generated silver nanoparticles. Journal of Agricultural and Food Chemistry, 61, 260–267. Mironenko, A., Modin, E., Sergeev, A., Voznesenskiy, S., & Bratskaya, S. (2014). Fabrication and optical properties of chitosan/Ag nanoparticles thin film composites. Chemical Engineering Journal, 244, 457–463. Ozin, G. A., & Hugues, F. (1983). Silver atoms and small silver clusters stabilized in Zeolite Y: Optical spectroscopy. Journal of Physical Chemistry, 87, 94–97. Pestov, A., Nazirov, A., Modin, E., Mironenko, A., & Bratskaya, S. (2015). Mechanism of Au(III) reduction by chitosan: Comprehensive study with 13 C and 1 H NMR analysis of chitosan degradation products. Carbohydrate Polymers, 117, 70–77. Potara, M., Gabudean, A. M., & Astilean, S. (2011). Solution-phase: Dual LSPR-SERS plasmonic sensors of high sensitivity and stability based on chitosan-coated anisotropic silver nanoparticles. Journal of Physical Chemistry, 21, 3625–3633. Rai, M., Yadav, A., & Gade, A. (2009). Silver Nanoparticles as a new generation of antimicrobials. Biotechnology Advances, 27(1), 76–83. Rajesh, R., Sujanthi, E., Kumar, S. S., & Venkatesan, R. (2015). Designing versatile heterogeneous catalysts based on Ag and Au nanoparticles decorated on chitosan functionalized graphene oxide. Physical Chemistry Chemical Physics, 17, 11329–11340. Reicha, F. M., Sarhan, A., Abdel-Hamid, M. I., & El-Sherbiny, I. M. (2012). Preparation of silver nanoparticles in the presence of chitosan by electrochemical method. Carbohydrate Polymers, 89, 236–244. Roberts, J. D., & Caserio, M. C. (1967). Modern organic chemistry. New York: W.A. Benjamin, Inc. Rodríguez-Argüelles, M. C., Sieiro, C., Cao, R., & Nasi, L. (2011). Chitosan and silver nanoparticles as pudding with raisins with antimicrobial properties. Journal of Colloid and Interface Science, 364, 80–84. Sanpui, P., Murugadoss, A., Prasad, P. V. D., Ghosh, S. S., & Chattopadhyay, A. (2008). The antibacterial properties of a novel chitosan-Ag-nanoparticle composite. International Journal of Food Microbiology, 124, 142–146. Singh, A., Narvi, S. S., Dutta, P. K., & Pandey, N. D. (2006). External stimuli response on a novel chitosan hydrogel crosslinked with formaldehyde. Bulletin of Material Science, 29, 233–238. Wang, W., & Cui, W. (2008). Chitosan-luminol reduced gold nanoflowers: From one-Pot synthesis to morphology-dependent SPR and chemiluminescence sensing. The Journal of Physical Chemistry C, 112, 10759–10766. Wei, D., & Qian, W. (2008). Facile synthesis of Ag and Au nanoparticles utilizing chitosan as a mediator agent. Colloids and Surfaces B: Biointerfaces, 62, 136–142. Williams, D. H., & Fleming, I. (1980). Spectroscopic methods in organic chemistry. Maidenhead: McGraw-Hill. Zhang, Y., Chang, G., Liu, S., Lu, W., Tian, J., & Sun, X. (2011). A new preparation of Au nanoplates and their application for glucose sensing. Biosensors and Bioelectronics, 28, 344–348.