Nanocomposites based on cationic polyelectrolytes and silver nanoparticles: Synthesis, characterization, molybdate retention and antimicrobial activity

2.1 Reagents

Silver nitrate (AgNO₃, Aldrich) was used as source of silver ions to carry out the synthesis of AgNPs, acrylic acid (99%, Aldrich) was used as stabilizing agent and sodium borohydride (99%, Aldrich) was used as reducing agent. 4-Chloromethylstyrene (Aldrich, 90%) and triethylamine (Aldrich, 99.5%) were used for the synthesis of N-vinylbenzyl-N-triethylammonium chloride using acetone as solvent (Merck, 99.5%). N,N-methylene-bis-acrylamide (99%, Aldrich) and ammonium persulfate (98%, Aldrich) were used as crosslinking agent and radical initiator, respectively. Ammonium molybdate was used for preparation of molybdate solutions. pHs of solutions were regulated using 0.1 mol/L of NaOH and HNO₃ solutions. BaCl₂ (Merck), sulfuric acid (Merck) and Agar Tryptone Soya (Aldrich) were used for test of antimicrobial activity. E. coli and S. aureus were used as microorganism models corresponding to Gram-negative and Gram-positive bacteria, respectively. Deionized water was used in all experiments.

2.2 Synthesis of polymerizable nanostructured crosslinker (PNC)

Synthesis of polymerizable nanostructured crosslinker (PNC) was performed by chemical reduction. For that, 45.0 mL of AgNO₃ (3.3 × 10⁻⁴ mol/L) and 3.0 mL of acrylic acid (0.02 molL⁻¹) were mixed and stirred for 10 min. Later, 2.0 mL of NaBH₄ solution were added to the mixture (1:5 Ag:BH₄⁻ ratio); then, mixture was stirred for 1 h at 100 rpm. Finally, PNCs were characterized by ultraviolet–visible spectroscopy (UV–vis, Shimadzu UV-1700, PharmaSpec) and dynamic light scattering (DLS, ZetasizerNano ZS90). Nanoparticles were concentrated using a centrifuge (Hettich Universal 320R) for 5 h at 5000 rpm and 15 °C; later, supernatant was eliminated by decantation and precipitate was diluted to total volume of 5.0 mL (Córdoba and Palencia, 2017).

2.3 Synthesis of N-vinylbenzyl-N-triethylammonium chloride

3.0 mL (19.2 mmol) of 4-chloromethylstyrene and 3.0 mL (21.4 mmol) of triethylamine were separately dissolved in 3.0 mL of acetone; later, solutions were mixed and 6.0 mL of acetone were added. The mixture remained in contact for 5 days at 25 °C. N-vinylbenzyl-N-triethylammonium chloride was washed with an excess of acetone to remove non-reacting precursors. Synthesis was verified by a polymerization test in water (Palencia et al., 2016a, 2016b). Reaction yield was gravimetrically determined. Finally, N-vinylbenzyl-N-triethylammonium chloride was characterized by 13-carbon nuclear magnetic resonance (¹³C NMR, Bruker 400 UltraShield).

2.4 Synthesis of nanostructured poly(N-vinylbenzyl-N-triethylammonium chloride) (NPVBtEA)

For the synthesis of nanostructured poly(N-vinylbenzyl-N-triethylammonium chloride) (NPVBtEA), N-vinylbenzyl-N-triethylammonium chloride (1.0 g), ammonium persulfate (29.9 mg) and N,N-methylene-bis-acrylamide (66.6 mg) were dissolved in 3.0 mL of aqueous dispersion of PNC previously synthesized. The mixture was reacted at 80 °C for 5 h in nitrogenous atmosphere in order to deactivate the polymerization inhibitor. In addition, non-nanostructured poly(N-vinylbenzyl-N-triethylammonium chloride) (PVBtEA) was synthesized at the same conditions used for the synthesis of NPVBtEA. Polymers were purified by diafiltration using a stirred-cell ultrafiltration unit (Amicon) and cellulose membranes (Millipore, 10 kDa); in addition, for NPVBtEA, remaining silver concentration was monitored in the permeate using atomic absorption spectroscopy (AAS, AA-7000 Spectrophotometer, Shimadzu). Later, polymers were dried using an airflow oven at 80 °C and, finally, polymers were ground and sieved (1–2 mm mesh).

Polymers were characterized by Fourier-transform infrared spectroscopy (FT-IR, Shimadzu, FT-IR 8400), 13-carbon nuclear magnetic resonance (¹³CNMR, Bruker 400 UltraShield), Surface-enhanced Raman scattering (SERS, ThermoFisher Scientific), thermogravimetric analysis (TGA, Q50-TGA TA Instruments), differential scanning calorimetry (DSC, Discovery DSC-25, TA instruments) and scanning electron microscopy (SEM, EOL/JEM 1200 EX II). In addition, swelling capacity (SC) of NPVBtEA and PVBtEA were measured by method of tea bag (Palencia et al., 2017a, 2017b), for that, tea bag with a known mass of polymer (0.1 ± 0.0001 g) was introduced into bi-distilled water (100 mL) for 24 h. After, excess of solution is eliminated by gravity for 10 min. A blank experiment in absence of polymer was performed. All experiments were performed by triplicate. Thus, SC is calculated by

2.5 Study of molybdate retention properties

Retention properties of NPVBtEA were studied using a batch-type reactor and MoO₄²⁻ as anion of interest. For that, NPVBtEA samples (50.0 mg) were individually placed in contact with metal ion solutions (100 mg L⁻¹) at different values of pH (5.0, 7.0 and 9.0) and ionic strength (0.0, 0.5 and 1.5% of NaCl). Concentration of Mo was monitored over time by AAS using a AA-7000 Spectrophotometer, (Shimadzu). Aliquots and calibration curves were diluted using a nitric acid solution (1%).

2.6 Evaluation of antibacterial activity

Antibacterial activity tests were performed using Tryptic Soy Broth (30 mg L⁻¹ using bi-distilled water as solvent) and two bacterial models: E. coli and S. aureus. Absorbance was measured at 625 nm and turbidity standards were prepared from barium chloride solution (0.048 mol L⁻¹) and sulfuric acid (0.180 mol L⁻¹). Colony-forming units (CFUs) were determined by McFarland scale using bacterial inoculums corresponding to 0.5 CFU mL⁻¹. Thus, 10.0 µL of bacterial dispersion was added to 5.0 mL of culture medium previously sterilized and contacted with a known mass of polymer (1.0 mg). Finally, system was incubated at 37 °C for 24 h. As blank was used NPVBtEA at the same conditions. In addition, two control experiments were performed using Ampicillin (0.50 g) as negative control and sorbitol (0.10 g) as positive control (Palencia et al., 2017a, 2017b).

3.1 Synthesis of polymerizable nanostructured crosslinker (PNC)

In the Fig. 1A is shown the UV–vis spectra over time for PNC dispersion. Surface plasmon resonance (SPR) with a maximum absorbance ∼400 nm can be identified. In general, SPR is considered an evidence of nanostructuration of silver atoms as a result of chemical reduction being NaBH₄ the reducing agent (i.e., Ag⁺ + 1e⁻ → Ag⁰) (Cañamares et al., 2008; Sanchez et al., 2002; Gou et al., 2016). SPR is produced by resonant oscillation of conduction electrons at the interface of nanometric particles stimulated by incident light (Cañamares et al., 2008); in addition, the change of maximum absorbance over time is shown in the Fig. 1B. It was concluded that AgNPs were stable during the monitored time (∼7 days). However, a decrease of maximum absorbance was observed between first two days.

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Fig. 1

(A) UV–vis spectra over time for PNC dispersion (from t₁ to t₇) (B) and change over time of maximum absorbance (from t₁ to t₇); (C) Dispersed light intensity in function of size for PNC by DLS and (D) illustration of NPC.

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Synthesis of AgNPs can be described by two stages, a first stage of nucleation where “seeds” are produced and a second growth stage or aggregation where Ag⁰ atom are transferred from aqueous phase to surface of nucleus, in addition, in this second stage, collisions between Ag⁰ clusters, among atoms and clusters can occur (Henglein and Giersig, 1999). According to Mie theory, if number of particles no changes during growth stage then position of plasmon band must shift to longer wavelengths and, therefore, an increase in the particle size is produced (Kolwas et al., 2009). Thus, as particle size is increased, particles are destabilized and precipitated producing a decrease of particle number in dispersion and a decrease in the intensity of maximum absorbance.

On the other hand, results of DLS are shown in the Fig. 1C. It can be seen that for PNC with lower size showed values of 5.62 ± 1.61 nm in diameter. However, for PNC with larger size, observed average diameter was 111.9 ± 74.49 nm.

Strategy used to synthesize the ENP was based on the obtaining of AgNPs stabilized by polymerizable monomers (i.e., acrylic acid). Thus, ENP is constituted by an inorganic phase (Ag⁰) and organic phase (vinyl monomer, see Fig. 1D). Thus, main difference between AgNPs synthesized in this work, respect to those reported by other researchers, is the stabilization using acrylic acid, which could permit the addition of AgNPs to polymer phase during free-radical polymerization. Stabilization of AgNPs by citric acid have been widely described (Parker, 1969; Zeng et al., 2008; Cheng et al., 2011), in consequence, since acrylic acid and citric acid are structurally very similar, it is suggested that when AgNPs are stabilized with acrylic acid, stabilization occurs analogously to that produced by citric acid. For synthesis of AgNPs using citrate two different chemical species have been identified and associated with bimodal distribution observed by DLS. The first one corresponds to silver citrate and the second one is associated to “free” Ag⁺ in the bulk of solution. When acrylic acid is used, bimodal distribution also is observed suggesting that two different chemical species of silver exist during the synthesis of nanoparticles by chemical reduction in aqueous solution.

3.2 Synthesis of N-vinylbenzyl-N-triethylammonium chloride

Synthesis of N-vinylbenzyl-N-triethylammonium chloride was based on the functionalization of 4-chloromethylstyrene. Reaction can be verified qualitatively by the formation of precipitate because an ionic product is formed in non-polar environment. N-vinylbenzyl-N-triethylammonium chloride was synthesized by the formation of one quaternary ammonium group via substitution of chlorine atom on 4-chloromethylstyrene, in consequence, a decrease of solubility in acetone is produced.

Reaction yield was 94%; in addition, respective ¹H NMR spectrum of ammonium quaternary monomer is shown in the Fig. 2. Ten signals can be identified in the spectrum: two correspond to the acetone that was used as solvent and non-deuterated chloroform and eight to the monomer. A triplet that integrates for nine protons is seen at 1.49 ppm that corresponds to the three equivalent methyl denoted as a, the methylenes b appear as a quartet at 3.47 ppm that integrates for six protons, a singlet corresponding to c is observed at 4.82 ppm. The most important signals appear at 5.39 and 5.83 ppm corresponding to the protons of the double bond g (J = 10.93 Hz) and h (J = 17.56 Hz), respectively, and proton f (J = 10.93 and 17.57 Hz) which appears as a double doublet and is seen at 6.72 ppm, indicating that double bond from 4-chloromethylstyrene is present and therefore the product is susceptible to be polymerized by free radical polymerization. Finally, the protons e and d of the aromatic ring appear as two doublets at 7.54 and 7.46 ppm.

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Fig. 2

¹³C NMR spectrum of N-vinylbenzyl-N-triethylammonium chloride.

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Synthesis of N-vinylbenzyl-N-triethylammonium chloride occurs by nucleophilic substitution in a single step (SN₂ reaction) since amines are very good nucleophiles and, under substitution conditions, amines form quaternary salts. An important factor that plays a role in this reaction is the character of the solvent; in consequence, the increasing stabilization of the nucleophile by the solvent produces a decreasing reactivity. Thus, polar protic solvents will stabilize the chloride ions through the formation of hydrogen bonds and, in consequence, formation of product couldn't be observed (Yeole et al., 2011).

3.3 Synthesis and characterization of NPVBtEA and PVBtEA

A comparison of characterization results between PVBtEA and NPVBtEA is shown below. Spectra of FT-IR and ¹³C NMR are shown in the Fig. 3A and B, respectively. A great likeness between PVBtEA and NPVBtEA spectra is observed, suggesting an evident result, polymerization was carry out correctly in both cases. From FT-IR spectra can be identified at 3360 cm⁻¹ the vibration of O—H bond, in particular, this signal is associated with acrylic acid of PNC and high hydrophilicity of polymers. Other signals are associated with CH₂, carbonyl group (on acrylic acid and N,N-methylene-bis-acrylamide structures) and C—N at ∼2950, ∼1650, ∼900 cm⁻¹, respectively. In addition, C⚌C vibration appears at ∼1620–1680 cm⁻¹.

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Fig. 3

(A) FTIR spectra of PVBtEA and NPVBtEA and (B) solid ¹³C NMR spectrum of PVBtEA and NPVBtEA.

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On the other hand, the following signal were identified from ¹³C NMR spectra: In the range from 30 to 55 ppm, signals are associated to methylene carbon; from 120 to 140 ppm, signals are associated with aromatic ring; from 80 to 100 and 140–180 ppm, signals are associated to C—N bonds resulting of functionalization of 4-chloromethylstyrene, and finally, also signals associated with crosslinking agent, N,N-methylene-bis-acrylamide, were identified: between 175 and 190 ppm for carbonyl group and between 75 and 90 ppm for N—C—N group.

Results of analysis by SERS of PVBtEA and NPVBtEA are shown in the Fig. 4. Two types of information can be extracted from these results. First, identification of functional groups in the samples, and second, characterization of interaction type between AgNPs and polymer in the NPVBtEA. Thus, characteristic signals of PVBtEA at 800, 1050, 1200 and 3000 cm⁻¹ corresponding to C—C (in the chain), ⚌C—H (in the plane) and C—H, respectively. At 1600 cm⁻¹ was identified the signal corresponding to C⚌O from crossliker used. On the other hand, it can be seen the enhancement of signals for NPVBtEA respect to PVBtEA for lower wavenumber.

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Fig. 4

SERS spectra of PVBtEA and NPVBtEA.

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SERS is based on the huge enhancement of the Raman emission of organic molecules when these are placed in the proximities of certain nanostructured metallic surfaces, in consequence, signal enhancement in the Raman spectrum can be used to identify the presence of AgNPs in the polymer phase; however, for it to occur, the metal nanoparticles must be relatively small in comparison with the wavelength of the excitation light (Kolwas et al., 2009; Mei et al., 2014). The successful application of metal nanoparticles in SERS strongly depends on the metal characteristics, in terms of morphology (shape, size and aggregation state) and the metal nature of the nanostructured metals.

Results of thermal analysis (TGA, DSC and differential thermal analysis, DTA, for PVBtEA and NPVBtEA) are shown in the Fig. 5. Significant changes in the thermal stabilities of PVBtEA and NPVBtEA were not observed and results are summarized in the Table 1. Thus, three degradation stages can be identified: In the stage 1, a mass loss around 7–8% at ∼100 °C can be explained for the release of adsorbed water from hydrophilic groups; in addition, two endothermic peaks are observed from DSC. In the stage 2, a degradation with loss of mass ∼22% is observed at 242 °C was attributed to loss of ammonium quaternary groups; and in the stage 3, decomposition of polystyrene segments was identified at 460–463 °C. However, a total degradation was not achieved (residual mass ∼12 ± 1%). It can be concluded that polymers, PVBtEA and NPVBtEA, were thermally stables until 242 °C. SEM images of PV tEA and NPV tEA are shown in the Fig. 6. It can be seen that materials are amorphous, porous and very similar morphology.

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Fig. 5

Thermal analysis of PVBtEA and NPVBtEA (TGA, DSC and DTA).

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Fig. 6

SEM images for samples of PVVtEA (A and B) and NPVBtEA (C and D).

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Swelling capacity of NPVBtEA compared with those of PVBtEA showed a small decrease (∼4%). Polymers absorbed ∼56 times their mass in water. Hydrophilic nature of ionic polymers is widely known and charged groups on polymer chain are the main factor to produce a favorable interaction with water molecules; however, when synthesis is carried out in the presence of PNC, the number of active sites, associated with ammonium quaternary groups, was not decreased by PNC added.

3.4 Molybdate retention by NPVBtEA

Effect of pH and ionic strength on molybdate retention, expressed as Mo retention, are shown in the Fig. 7. It can be observed that, at low ionic strength, retention decrease with the increase in pH (retentions decreases from ∼80 to ∼55% for values of pH from 5.0 to 9.0). However, as the ionic strength increases, a significant effect of pH on molybdate retention was not observed. However, the increase in the ionic strength produced a significant decrease of molybdate retention.

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Fig. 7

Mo retention curves over time at different pH values (5, 7 and 9) and ionic strength (0.0, 0.5 and 1.5% NaCl).

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Though molybdate retention properties of NPVBtEA have not been previously reported, it is expected a similar behavior to that observed for PVBtEA because it can be assumed that nanostructuration no produces effect on retention. Thus, ammonium quaternary groups are the main interaction sites available to interact with molybdate ions. It has been reported that interaction of ammonium quaternary groups and molybdate ions is mainly by electrostatic interaction; however, since NPVBtEA absorbed water, another possible interaction is the inclusion of ions in the polymer phases by water flux. For electrostatic interaction, assuming that all ammonium quaternary groups on polymer chains are all the same, it is expected that interaction is weakened by the presence of ions in solution because they decrease the effective electric field of the interacting charges.

3.5 Evaluation of antibacterial activity

The results of evaluation of antibacterial properties of PVBtEA and NPVBtEA against E. coli and S. aureus are shown in the Fig. 8. It can be seen that inhibition percentage for NPVBtEA is 100% using NPVBtEA. The increase in the antibacterial activity of NPVBtEA is explained by Ag⁰ in the PNC. It has been reported that minimum inhibitory concentration of AgNPs is 50 μg/mL (Maiti et al., 2014). In addition, for silver ion concentration of 0.2 mg/L produce a total inhibitory effect (Feng et al., 2000; Jung et al., 2008).

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Fig. 8

Inhibition percentage of PVBtEA and NPVBtEA against E. coli and S. aureus.

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The mechanism of the antibacterial activity of AgNPs is complex (Habiba et al., 2015), some authors have associated the high activity of AgNPs to the capability of these to act as a reservoir of silver ions that are released into the bacteria, interacting with the thiol groups of enzymes and proteins producing the inhibition of their functions (Shrivastava et al., 2007). Other authors believe that nanoparticles can modulate signal transduction between bacteria stoping their growth, but also, other authors have proposed that the formation of free radicals by nanoparticles can damage the cell membrane and make it porous (Habiba et al., 2015; Shrivastava et al., 2007; Palencia et al., 2015).