Hybrid graphene oxide-based silver nanoparticles hydrogels as potential adsorbents for dye wastewater

treatment Ana Catarina Ferreira Cajada Instituto Superior Técnico, Lisbon

November 2016

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Abstract

This work focuses on the synthesis and characterization of graphene-based hydrogel containing silver nanoparticles (AgNPs) with the aim of studying their swelling and dye adsorption capacities in view of their potential application in wastewater treatment. Hydrogels have been widely studied due to their improved physicochemical properties and used as antibacterial platforms, wastewater pollutants adsorbents and drug deliverers. The synthesis of hybrid hydrogels aims to combine the components’ abilities in order to obtain a material with improved synergic properties. Graphene oxide (GO) adds a wide surface area as well as a good mechanical flexibility to the hydrogel while AgNPs contributes with its hardness and biocompatibility. The hydrogels’ structure was characterized using spectroscopic and microscopic techniques. Furthermore, interactions between the hydrogel, graphene oxide and the silver nanoparticles were studied.

As expected, the combination the above mentioned elements led to a hydrogel with enhanced swelling and adsorption capacities towards wastewater common dyes such as rhodamine 6 G and 2-nitrophenol.

Keywords: hydrogel, graphene oxide, silver nanoparticles, swelling, dye adsorption

1. Introduction The synthesis of hydrogels composed by polymers has been growing in the past few years, as they are materials with a wide range of qualities such as biocompatibility, elasticity, swelling capacity, network characteristics and environmental sensitivity. Polymeric hydrogels additionally show a responsive behavior to changes in their surroundings, such as temperature, pH, salt concentrations, even the ability to store and restrain functional groups. These structures exhibit improved physicochemical properties endowed with several applications in different fields such as tissue engineering, agriculture, regenerative medicine, light harvesting and hybrid composites. Despite all the aforementioned advantages, the traditional hydrogel still has disadvantages regarding their properties and usage. Its mechanical properties are still poor compared to other materials and they have small equilibrium-swelling ratio besides of having limited functional properties [1–5].

Graphene oxide (GO) has been attracting attention due to its excellent electronic and optical properties, large surface area, mechanical flexibility, etc [6][7][8]. The graphite when oxidized

to GO (Figure 1), gains several functional groups in its surface and edges such as hydrophilic oxygen containing groups like carboxyl, carbonyl, epoxy and hydroxyl groups, which will allow GO nanosheets to interact more strongly with other molecules on its surroundings, through electrostatic forces and hydrogen bonding [1][8–10].

Figure 1 Graphene and graphene oxide molecular structures. Taken from [11].

Graphene and GO have been used to synthesize 1D carbon nanotubes, 2D layer films and 3D gels in nanomaterial research. Graphene based hydrogels attracted attention due to its excellent characteristics, in particular, mechanical strength, large surface and adsorption capacity. Therefore, the combination of graphene with several macromolecules and polymers gained considerable attention and has been used to synthesize hybrid hydrogels with tailored

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properties with a wide range of applications [1][4][9].

The combination of graphene-based materials with metal nanoparticles has received special attention in recent years due to the possible synergistic effect, which endows these materials with a wider range of applications [1][11].

Silver nanoparticles (AgNPs) have been used in several fields due to their physical and chemical properties. They have potential applications in fields like nanotechnology, nanomedicine and bio-applications: antibacterial, anti-inflammatory, anti-cancer agents, diagnostics, drug delivery, etc [12][13]. AgNPs seem to be good alternative antibacterial agents to antibiotics and have shown to have the ability to overcome the bacterial resistance against antibiotics mainly due to characteristics like their large surface-to-volume ratios and surface structure. They have already shown to be active against yeast and 16 species of bacteria including Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa [14–16].

Nanoparticles have been incorporated inside a gel network in order to prepare gel-nanocomposites where the its individual properties would remain intact as well as a synergy of the properties from both components would be possible [11]. Thus, besides adding an antibacterial feature to the hydrogel, the incorporation of nanoparticles can improve the strength of the hydrogel and also proved to increase its rate and adsorption capacity [17].

GO-based hydrogels have shown to provide an effective way to separate and remove pollutant dyes from water, plus different weight retention values by the composites were reported [9]. Several groups reported potential dye removal application of GO-based composites such as PVA and chitosan hydrogels. The most commonly dyes used to assess the adsorption capacity of these materials are usually very photostable and with specific properties, such as methylene blue, rhodamine B, rhodamine 101, 4-nitrophenol [9][11][18][19].

In this work, PAM-GO hydrogels were synthesized and their ability to adsorb dyes (rhodamine 6G and 2-nitrophenol) from aqueous solutions was tested under distinct environmental conditions in an attempt to obtain an improved vehicle for

wastewater treatment. Further contribution to this goal was sought by the addition of silver nanoparticles to the hybrid hydrogel. The latter system was tested further for its antibacterial efficiency against pathogenic bacteria with clinical relevance in the second part of this work.

2. Materials and Methods

2.1. Materials

Graphite powder used for graphene oxide synthesis was from Cymit Química SL. Sulfuric acid (96%), sodium borohydride and absolute ethanol from Carlo Erba. Hydrochloric acid (37%), hydrogen peroxide (30%), chitosan (Mw=110,000-150,000; high purity), polyethylene glycol (PEG, average Mn 4,600), polyethylenimine (PEI, high molecular weight, water-free) were acquired from Sigma Aldrich. Potassium permanganate was obtained from Analak. Pure glacial acetic acid from Epenhuysen Chemie. For polyacrylamide hydrogel synthesis the acrylamide was from E. Merck, silver nitrate from RP ACS, the crosslinker N,N′-Methylenebisacrylamide (MBA, 99%) from Fluka Chemika and potassium persulphate from Merck. At the pollutants intake tests acridine orange (AO) and Rhodamine-6-G (R6G) available on laboratory were used as well as 2-Nitrophenol purchased from The British Drug Houses LTD. Disodium hydrogen phosphate anhydrous obtained from Fluka Chemika and citric acid from Sigma Aldrich were used for citrate-phosphate buffer. All other chemicals were used as received without further purification.

2.2. Graphene oxide synthesis

Method 1 Graphite powder (1g) was dissolved in H2SO4 concentrated (23 mL) in an Erlenmeyer (250 mL). The solution was agitated during the night for 12h. The temperature of the recipient was lowered to 0ºC prior slow addition of 3g of KMnO4. The mixture was agitated for 30 minutes and subjected to 3h of ultrasounds. 46 mL of distilled water under agitation and the solution was heated for 30 minutes. After cooling down, 140 mL of distilled water was added to stop the graphite oxidation reaction and the solution was agitated for approximately 1 hour. While agitating H2O2 (10 mL) was added drop by drop. Method 2 This method followed the same steps previously described for the first graphene oxide synthesis method. After the addition of KMnO4 the mixture was sonicated for a period of 6h. Subsequently the mixture was heated and brought

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to boiling for 30 minutes. After cooling H2O2 was directly added, skipping the water addition step. As the preceding method, the mixture was subjected to several purification steps, consisting in washing with DW and ethanol, and centrifugation phases (cycles of 5000 rpm) [20–22].

2.3. Silver nanoparticles synthesis

Chitosan 4 mL of a AgNO3 solution (52.0 mM) was added into a chitosan solution (6.92 mg/mL) and were stirred until a homogenous mixture was obtained. The mixture was heated for 12 h at 95 °C. The color of the solution progressed from colorless to light yellow after a couple of hours under heating [23]. PEI with NaBH4 1 mL of a AgNO3 solution (5.0 wt%) was added to 3 mL of PEI solution (min. 0.02 wt%) and stirred for one hour. To accomplish the silver reduction NaBH4 was added (NaBH4/ AgNO3=0.75). The mixture was agitated at room temperature for 3h in order to complete the reduction [24][25].

2.4. GO hydrogel synthesis

PEI In order to synthesize PEI-GO hydrogel several GO concentrations were tested: 5.0 and 7.5 mg/mL; And PEI 0.5; 0.75 and 1.0 mg/mL. First, GO was dissolved in DW and subjected to ultrasounds. Two addition sequences were tested: first GO and then PEI, and the other way around. The second sequence seems to favor the formation of GO hydrogel rather than first. No agitation is necessary to form the hydrogel. After formation, PEI excess was removed. Chitosan Stock solution of chitosan in DW (0,05% w/v) with hydrochloric acid 0.008% (w/v). 0.5 mL of a GO dispersion (10 mg/mL) was mixed with 0.1 mL of chitosan, total volume of 1.0 mL. Addition order: chitosan, DW and GO. Gelation only happens without agitation [26]. Polyacrylamide (PAM) For 10 mL of DW, 1.0g of acrylamide was dissolved in DW and stirred for some minutes, followed by the addition of 7.0mg of MBA. To help with polymerization, AgNO3 (2.0 g) was added prior adding 11 mg of potassium persulfate (KPS). The polymerization takes thereabout 1 minute to happen. When using temperature as polymerization precursor, a recipient with the final mixture is placed in a water bath at 65ºC for 30 minutes in order to get the hydrogel. To synthesize hydrogel with GO (PAM-

GO), the same steps are followed but the initial solution contains GO (2.5 mg/mL) [27].

2.5. Hydrogel synthesis with AgNPs

In situ Both PAM and PAM-GO hydrogels, synthesized as previously described, were deposited in a falcon containing 10 mL of NaBH4 so the reduction of AgNO3 would take place. The hydrogels were left in the reducer solution for 24h at 4ºC [25][28][29][30]. Chitosan previously synthesized AgNPs were used in the AgNP-hydrogel synthetic process. For 2.0 mL of total volume, 1.0 mL of Chitosan AgNPs was added to the solution of PAM hydrogel. Regarding PAM-GO hydrogel, in 2.0 mL, GO was dissolved in 1.0 mL of DI water and subjected to 30 minutes of ultrasounds. Subsequently, the silver nanoparticles were added followed by the remaining components. PEI with NaBH4 The same method was followed to synthesize hydrogel with PEI-NaBH4 silver nanoparticles.

2.6. Swelling ratio

Swelling studies were made to determine the amount of buffer absorbed through time by the hydrogels, for that, the as-prepared hydrogel discs were weighted before the process of dye adsorption and after 24 or 30 hours of contact with the dye [2][31][32].

𝑆𝑅 =𝐷%& − 𝐷%(

𝐷%(∗ 100%

Equation 1 Swelling ratio of the hydrogel.

Where SR is the swelling ratio, Dwt the weight of the hydrogel disc after t time of absorption and Dw0 is the initial weight, before starting the swelling test.

2.7. Dye pollutant intake

The hydrogel discs were immersed in 12 mL of Rhodamine 6 G (R6G, 5.0-8.0 μM) and 2-Nitrophenols (2-NP, 0.01 mM), separately, in citrate-phosphate buffer at four pH values: 2.0, 4.0, 5.0 and 7.4. Several samples were collected during a period of time until approximately 30 hours. Dye removal was calculated using Lambert-Beer equation, with the absorbance measured at 529.5 nm for R6G, wavelength at which the extinction coefficient is known (eR6G=116,000 M-1cm-1). For 2-NP only the absorbance values were used.

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𝐷𝑅 =𝐴( − 𝐴l𝐴(

∗ 100%

Equation 2 Dye removal.

Where DR is the dye removal, A0 is the absorbance of the dye stock solution at the respective test pH and Al is the absorbance at maximum wavelength of the samples collected.

2.8. Instrumentation

Besides the equipment showed below, equipment like HERMLE centrifuge and ultrasounds Elma S 30H Elmasonic was used during preparation and purification processes. UV-visible absorption spectra were recorded on Jasco V-560 and PerkinElmer spectrophotometer Lambda 35. The range used was 700 to 200 nm and typically using a bandwidth of 1 or 0.5 nm. Ground-state diffuse reflectance absorption spectra (GSDR) were measured using a homemade diffuse reflectance laser flash photolysis setup, with a 250 W W-Halogen lamp as monitoring lamp. Steady-state fluorescence spectra were obtained with a SPEX Fluorolog spectrofluorimeter (HORIBA JobinYvon) in a FL3–11 configuration. Quartz cells with an optical length of 1 cm were used. Fluorescence lifetime imaging microscopy (FLIM) was performed using the time-resolved confocal microscope Microtime 200 set-up from PicoQuant GmbH equipped with an Olympus IX 71 inverted microscope and Fluorescence correlation spectroscopy (FCS) was used to estimate the hydrodynamic radius of the distinct synthesized AgNPs. The confocal FCS setup is identical to FLIM, a more detailed information can be found elsewhere [8].

TEM images were obtained with a Hitachi H-8100 electron microscope operated at 200 kV. Scanning electron micrographs (SEMs) were obtained in MicroLab facility at IST through an analytical FEG-SEM: JEOL 7001F with Oxford light elements EDS detector and EBSD detector. The experimental zeta potential values (ζ) were determined in a Doppler electrophoretic light scattering analyzer, Zetasizer Nano ZS from Malvern Instruments Ltd.

3. Results and Discussion

3.1. Characterization

The GO synthesis, as well as its purification, were followed by different spectroscopic methods as UV-Vis absorption, steady state fluorescence (lexc=400 nm) and Raman. Its morphology was observed by TEM and FLIM. Also, zeta potential was measured to assess the level of oxidation of both synthesized GO samples.

Figure 2 UV-Vis absorption spectra of GO synthesized by method 1 and 2. [GO]=0.1 mg/mL.

Besides the intensity difference between both GOs absorption spectra (Figure 2), they both present the typical maximum at around 231±2 nm and the shoulder around 303±2 nm, corresponding to the π→π∗ transition of the C–C and C=C bonds and the n→π∗ transition of the carbonyl bonds in sp3 hybrid regions, respectively, confirming their oxidation. GO2 presents a red shift comparing to GO1, which results from the higher number of conjugations in the former, leading to an increase of the λmax of the molecule. The better defined peaks obtained for the GO1 sample seem to indicate a better solvent dispersion and/or smaller flakes in the case of the former which introduces less scattering in the detected signal. Regarding fluorescence spectra, GO2 showed a less intense photoluminescence confirming the less oxidation of the sample. Additionally to what was said above about the sample dispersion, a less-well dispersed sample with multilayers, would lead to a quenching of the luminescence signal of the sample [33]. A possible reason for the less oxygenation in GO2 could come from the fact that higher temperatures were used and/or that an excess of H2O2 was used during its synthesis, thus originating reduced graphene oxide [11]. Large graphene oxide sheets were observed with wrinkled structures randomly aggregated in both GO TEM images. Also, darker zones on the images indicate the presence of a higher number of GO nanosheets, demonstrating the existence of several stacked layers.

0

1

2

200 300 400 500

Absorban

ce

Wavelength,λ (nm)

AGO10.1mg/mL

GO20.1mg/mL

0

500

1000

1500

2000

850 1350 1850

Intensity

(a.u.)

Ramanshift(cm-1)

B GO1

GOr

GO2

5

Figure 3 TEM images of GO1 (A) and GO2 (B) samples. [GO1] =0.1mg/mL, [GO2] =0.05 mg/mL.

Raman spectroscopy was performed in order to highlight any possible difference between the GO samples synthesized, as it allows us to study the bonding nature of graphite materials, crystallinity and the disorder on GO [7][22]. A red shift is observed in both bands when comparing GO2, 1603 and 1352 cm-1, to GO1, 1587 and 1346 cm-

1. Even though none of the peaks are the same for any GO type tested, the values obtained are very similar to the ones presented in the literature [7][9][22][34].

For zeta potential measurements the concentration used was 1.0 mg/mL and the dispersions had a pH around 5. It is known that, due to the ionization of its carboxylic and phenolic hydroxyl groups, graphene oxide has a very negative zeta potential. Its decrease is a consequence of the removal of functional groups after the reduction reaction [22][35]. Inside the range of pHs used in the reference, for our working pH, the graphene oxide zeta potential it’s approximately -25 mV, which is in accordance with the results obtained for the GO samples synthesized (-27 mV) [35].

The formation of silver nanoparticles was followed using UV-Vis spectroscopy and controlled by observing the emergence of the surface plasmon surface resonance (SPR) band around 400 nm. For the methods used for nanoparticles synthesis, the SPR band was observed at different wavelengths [25][29][30].

It has been shown in literature that different wavelengths correspond to different particle sizes, diameters and morphologies, both influenced by the synthesis protocol, such as the choice of the type of solvent used or the amount of the initial AgNO3 [25][36]. The chitosan-based nanoparticles (CS-AgNPs) have the highest wavelength with the SPR band at 414 nm while PEI-NaBH4 AgNPs has it at ~400 nm. A red shift in the SPR band on the absorption spectrum follows the increase of the nanoparticle’s size,

thus the results here obtained are coherent with the mentioned in literature [36].

Figure 4 TEM images of CS-AgNPs (A) and PEI-NaBH4 AgNPs (B). UV-Vis absorption spectra of AgNPs (C) and FCS autocorrelation curves for AgNPs (D).

TEM images of the silver nanoparticles show mostly spherical nanoparticles with diameter of approximately 32 and 16 nm for CS-AgNPs and PEI-NaBH4 AgNPs respectively. FCS measurements were performed for AgNPs dispersed in DW. The hydrodynamic diameters obtained from applying the Stokes-Einstein equation (using the diffusion coefficient retrieved from the autocorrelation curves) were dH=22 nm and 47 nm, respectively, for AgNPs with PEI-NaBH4 and Chitosan. The fact that the sizes obtained for the AgNPs using FCS are larger than those obtained by TEM can be understood knowing that in FCS we measure the size of a sphere diffusing in a solvent plus solvent molecules that move along that sphere.

3.2. Interactions with GO

The interaction between GO and the reactants involved in AgNPs synthesis was studied recurring to UV-Vis absorption and fluorescence spectroscopy. The concentration of GO was kept constant (0.1 mg/mL) during the measurements, being the concentration of PEI, Chitosan or any other component modified. PEI showed to have a quenching effect on GO. GO photoluminescence maximum around 580 nm ceases to exist upon addition of the polymer. The luminescence quenching of GO by PEI can be examined using a Stern-Volmer (SV) plot. This quenching effect is much less pronounced in the case of GO2 which has a very weak fluorescence itself. The effectual quenching originates from the combination of hydrogen bonding and electrostatic interactions between amines in PEI and GO sheets, which promote quenching probably via an efficient

B A

B. d≈16±8 nm A. d≈32±5 nm

0

0.25

0.5

300 400 500 600

Absorbance

Wavelength,λ (nm)

C ChitosanAgNPsPEI-NaBH4AgNPs

0

0.5

1

1.5

2

2.5

0.01 0.1 1 10 100

G(t)

time(ms)

D AgNP-PEI-NABH4AgNP-Chitosan

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charge transfer process. The quenching efficiency will then depend on the nature of the oxygenated group in GO. Similar results were observed upon the addition of chitosan to the GO suspensions. Concluding, both polymers used for silver nanoparticles synthesis present a high quenching of GO photoluminescence.

Figure 5 Fluorescence spectra of GO1 and PEI(0.05, 0.1 and 0.25 mg/mL). Inset: SV plot for PEI with GO1 and GO2. [GO]=0.1 mg/mL;λexc=400 nm.

After testing the interactions between PEI and Chitosan with the GO, the interactions between the last and the different types of silver nanoparticles synthesized were tested. The addition of nanoparticles to the GO suspension did not lead to any specific change in the absorption spectra but it leads to a quenching of GO luminescence. In general, metal nanoparticles are efficient quenchers of many emitters such as dyes and polymers through a mechanism that may involve charge transfer or non-radiative energy transfer. The PL quenching of GO had already been reported for GO modified with AgNP which was used for detecting different biological species [37].

3.3. Hydrogel

The hydrogel samples used for characterization of PAM hydrogels consisted in small circular discs with approximately 7 mm diameter, containing 200 μL of the solution used to synthesize PAM hydrogel, as described in Material and Methods section. Hydrogel characterization was done using ground-state diffuse reflectance (GSDR) for samples of PAM and PAM-GO hydrogels synthesized with AgNO3 as polymerization enhancer. None of the hydrogels synthesized either with PEI or Chitosan had the right consistency or shape to be subjected to GSDR. In the hydrogel, the silver salt, the silver ions get adsorbed in its network by ion exchange interactions with the hydrogels’ functional groups. These interactions may be enhanced by the presence of graphene oxide, originating a more

robust hydrogel with improved properties. The carboxyl and hydroxyl groups existing on the GO’s surface interact with the amide groups of PAM by forming strong hydrogen bonds [9][38][31]. GSDR spectrum was used to confirm the presence of silver nanoparticles within the hydrogel matrix. In situ synthesis of silver nanoparticles by reduction of AgNO3 within a GO-based hydrogel was successfully achieved.

Figure 6 GSDR spectra of PAM (A) and PAM-GO (B) hydrogel: control and with silver nanoparticles synthesized in situ reduced with NaBH4.

For morphology tests SEM images were recorded for different sets of hydrogels with different compositions. Control samples were PAM and PAM-GO hydrogels and the test samples were the two types of hydrogels containing nanoparticles, CS and PEI-NaBH4 AgNPs. SEM images also allowed verifying the presence of preformed silver nanoparticles within the hydrogels both in the absence or in the presence of GO. Quite distinct images of the surface are obtained upon the addition of preformed AgNPs to PAM hydrogels. A good coverage of the latter with well-dispersed AgNPs is obtained with sizes that are within the range of data previously obtained by TEM and FCS (see above).

Figure 7 SEM images of PAM (up) and PAM-GO (down) PEI-NaBH4 AgNPs hydrogel at different magnifications.

0.0E+00

1.0E+07

2.0E+07

3.0E+07

450 550 650

INT/OD 4

00nm

(a.u.)

Wavelength,λ(nm)

GO1(0.1mg/mL)GO1+PEI(0.05mg/mL)GO1+PEI(0.1mg/mL)GO1+PEI(0.25mg/mL) 1.0

4.0

7.0

0 0.1 0.2F 0/F

[PEI](mg/mL)

GO1 GO2

0

0.1

0.2

300 550 800

Absorban

ce

Wavelength,λ (nm)

A ControlPAMPAM+AgNPsPAM+AgNPs2

0

0.025

0.05

300 550 800

Absorban

ce

Wavelength,λ (nm)

B GO1+AgNPs1GO1+AgNPs2GO1+AgNPs3ControlPAM-GO

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In PAM-GO hydrogels containing nanoparticles in the network, some long and thin 3D porous nanostructures can be observed. These nanostructures are the network structures of the hydrogel where some silver nanoparticles can deposit. Similar results were reported in a study with PAM-PEG hydrogels also containing silver nanoparticles [30]. The spherical silver nanoparticles are visible and can be observed throughout the surface of the hydrogel. The size of the PEI-NABH4 based nanoparticles observed correspond to the diameter previously acquired with TEM and FCS, approximately 20 nm.

When synthesizing the hydrogels with CS-AgNPs, it was observed that no temperature was needed for the hydrogel to be formed. 2-3 minutes after making the mixture with the acrylamide, CS-AgNPs, MBA and KPS, the hydrogel would form itself without temperature aid. Concluding, chitosan may have played an important role in the polymerization process of the hydrogel, making unnecessary the use of temperature.

FLIM images of the produced hydrogels were obtained using the fluorescence of R6G adsorbed to these structures. In the absence of the adsorbed dye no signal could be detected from the PAM hydrogel. In the absence of GO, it is possible to detect a very porous surface for the PAM hydrogel, consistent with SEM and literature data [39]. The presence of preformed AgNPs, in both cases studied, lead to a decrease of the long fluorescence lifetimes, which change from 3.5 ns peak to 3.0 and 2.3 ns respectively, for PAM-GO with CS-AgNPs and with PEI-NaBH4 AgNPs, slightly shorter than the value obtained for R6G in water (~4.0 ns taken from [40]). In the case of the latter, a second lifetime population appears centered at 1.3 ns. This clear decrease in fluorescence lifetime, which is followed by an increase in fluorescence intensity can be an indication of MEF.

Figure 8 FLIM images of PAM (A) and PAM-GO PEI-NaBH4 AgNPs (B) and average fluorescence lifetime distribution (C) of PAM (grey line), PAM-GO (black line), PAM-GO PEI-NaBH4 AgNPs (Û). B’ is the corresponding intensity image of B. Scale bar represents 10 µm (A) and 4 µm (B).

3.4. R6G: Swelling and adsorption

One of the applications of hydrogels is dye removal from wastewaters using as procedure the adsorption of the dye to its network. For the absorption of the solution, the following percentages of swelling of the control discs, PAM and PAM-GO, were obtained.

Table 1 Swelling ratio for control PAM and PAM-GO hydrogels for pH2,4,5 and 7.4.

Swelling ratio (%) PAM PAM-GO

pH2.0 253.1 180.0

pH4.0 228.4 182.2

pH5.0 263.4 151.6

pH7.4 226.2 148.3

The solution pH does not seem to influence significantly the swelling capacity of either PAM or PAM-GO hydrogels within the pH range studied (<20%). Additionally, it can be observed that the addition of GO to the hydrogel leads to a decrease of its swelling capacity at every pH tested. These values are in accordance with other results obtained for PAM-GO hydrogels swelling capacities at the work pH values [41]. It is known that the swelling properties of the hydrogels depend mainly on the effective cross-link density of the hydrogels. So, GO could act as an additional cross-linker, thus increasing the cross-link density and therefore, leading to the reduction of the swelling ability of the hydrogel. Strong hydrogen-bonding interactions take place between the hydroxyl and carboxyl groups of GO nanosheets and the polymer chains, forming a more compact and robust network, leading to an enhancement of hydrogel’s mechanical strength, confirmed by the SEM images, and diminishing the swelling capacity [2][26][31].

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Normalized

freq

uency

Averagelifetime/ns

A

B

B’

C

8

Subsequently, and analogous to control hydrogels, the swelling ratio was calculated for PAM and PAM-GO hydrogels containing AgNPs. For these the swelling ratios were calculated using discs’ weight after 24h of buffer-dye absorption instead of 30h.

Table 2 Swelling ratio for all types of PAM hydrogels at pH4.0 and 7.4.

Swelling ratio (%) PAM PAM-GO

pH4.0 pH7.4 pH4.0 pH7.4

Control 228.4 226.2 182.2 148.3

CS-AgNPs 96.6 97.8 261.2 283.6

PEI-NaBH4 AgNPs 97.7 91.1 213.1 214.3

The discs were not synthesized all in the same occasion. The conditions were tried to be kept the same during all the synthesis processes, i.e. composition ratios, temperature, time, etc. Nevertheless, the control on all these parameters is not total, wherefore, some of the results may be affected by it. Also, due to the hydrogels’ different composition, the synthesis method had some alterations, mainly resulting in different hydration states of the hydrogels prior adsorption.

The decrease of the swelling ratio associated with the addition of GO to the hydrogel was compensated with the enhancement of its mechanical properties, which lead to a more efficient use of the hydrogels. On the other hand, the addition of the nanoparticles to the graphene-based hydrogel allowed not only to recover the initial swelling capacity of the hydrogel but also to enhance this capacity. The incorporation of silver nanoparticles, as well as the GO, increased hydrogel’s mechanical properties [17].

For PAM-GO CS-AgNPs hydrogels, the difference of swelling shown between both pH values is caused by the charges present in within the hydrogel. At a more basic pH, the molecules experience an increase of the repulsion forces between them since chitosan, as well as GO, is negatively charged at the given pH (chitosan pKa 6.5). This results in a wider network into the hydrogel allowing it to absorb a higher amount of aqueous solution [26].

The removal efficiencies of dyes, initial and final concentrations of R6G, cationic dye, and 2-nitrophenol, anionic dye, solutions, were determined by UV-Vis absorption intensity,

following the changes at their maximum absorption wavelength.

GO is negatively charged inside the hydrogel at both pHs under study considering the negative values of the zeta potential, so, the cationic dye adsorption is induced by multiple interactions: electrostatic interactions, π-π interaction and hydrogen bonding [2][18][31].

Table 3 Dye removal percentages of R6G for all the hydrogels synthesized.

DR (%)

PAM PAM-GO

Control CS-

AgNPs

PEI-NaBH4 AgNPs

Control CS-

AgNPs

PEI-NaBH4 AgNPs

pH4.0 70.3 11.0 80.7 91.9 31.1 77.7

pH7.4 69.7 32.4 79.8 86.1 54.5 75.5

The charge on the inside of the hydrogel at a particular pH determines the kind of interactions occur between the hydrogel (i.e. the GO and the AgNPs) and the dye molecules [17]. The presence of GO in the hydrogels network results in an increase of the its surface area, thus improving the drug loading ratios of the hydrogels. The increase in the adsorption capacity may be due to the interactions between the hydroxyl and carboxyl groups of GO and the PAM chains, which provide better load transfer between the PAM matrix and GO nanosheets. Therefore, the presence of GO in the PAM-GO network modifies the hydrophilic character of the hydrogel, producing a change in its affinity for the dye [2][41].

An accentuated decrease of R6G removal by CS-AgNPs containing hydrogels can be observed in the Table 3, especially at pH4.0 (approx. 85% decrease). This decrease may be due to the fact that at acid pH’s chitosan shows a cationic behavior (pKa=6.5 [26]), which means that the CS-AgNPs can easily bind to GO by anion-cation interactions, and when inserting R6G, also a cationic compound, they are both competing to interact with GO. In the case of hydrogel with PEI-NaBH4, even though the difference among them is not significant (~7%), the results obtained indicate that GO does not have a significant role in these hydrogel’s adsorption ability. Regarding the influence of pH, it can also be discarded in this case, since the results obtained for both pH values differ in 1 – 3%. The excellent rates of adsorption of PEI-NaBH4 containing hydrogels are attributed to chemical adsorption through strong pi–pi stacking and anion–cation interactions, as well as

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the large specific surface area and porous structure of the hydrogel.

During the dye removal tests, R6G absorption spectra showed a blue shift (~6 nm) at its maximum wavelength peak after contact with every hydrogel except for the ones containing CS-AgNPs. These silver nanoparticles seem to have a stabilizing effect of the rhodamine, both in PAM and PAM-GO.

To understand adsorption kinetics for the adsorption of R6G the experimental data was fitted applying a pseudo-second order model [17].

𝑡𝐷𝑅

= 1

𝑘1𝐷𝑅2341 +𝑡

𝐷𝑅234

Equation 3 Pseudo-second order adsorption kinetics.

Where DR is the dye removal (% dye removed), DRmax the maximum dye removal and k2 the pseudo-second order constant (min-1.% dye removed-1). The values of DRmax and k2 can be obtained from the slope and intercept of plots of t/DR vs. t, respectively.

Table 4 Kinetics parameters for the adsorption of R6G onto PAM and PAM-GO hydrogels.

PAM PAM-GO

pH DRmax

(% Dye removed)

K2 (min-1.% Dye removed-1)

DRmax (% Dye

removed)

K2 (min-1.% Dye removed-1)

2.0 73.0 1.3x10-4 92.6 2.1x10-4

4.0 70.9 9.5x10-5 92.6 2.2 x10-4

5.0 84.7 3.4 x10-5 90.9 2.2x10-4

7.4 70.4 1.0x10-4 86.2 2.4x10-4

For k2, the higher the value, the faster occurs the adsorption process of the dye to the hydrogel’s network. The fastest adsorption, according to the parameters calculated for a pseudo-second order kinetics, happens for both hydrogels at pH2.0. An enhancement of both adsorption capacity and rate is observed with the addition of GO to the hydrogel. Concluding, a graphene oxide-based hydrogel has a higher adsorption capacity and this adsorption additionally happens at a higher rate.

For the hydrogels containing silver nanoparticles an increase on the kinetic parameters was observed. In the case of PEI-NaBH4 containing hydrogels, for pH4.0, the pseudo-second order constant is around 1x10-3 what supposes an increase of the dye’s adsorption rate. For CS-AgNPs, the rates are in the same magnitude order

as the obtained for the control hydrogels (Table 4), showing that no improvement on the dye adsorption capacity was obtained by adding these AgNPs.

Release tests from the dyed hydrogels were also done, nevertheless no significant release of R6G was observed (between 1 - 4%), neither of any other component, for any of the hydrogels.

4. Conclusion In summary, the hybrid hydrogels synthesized displayed good mechanical stability and toughness. This conclusion is based on the decrease of the swelling capacity of the hydrogels when adding GO to its network, due the strong interactions between the hydrophilic groups of GO and the polymer. However, the swelling capacity of the hydrogel is compromised when adding GO in favor of a more robust hydrogel. Despite that, the addition of AgNPs to the graphene oxide-based hydrogel results in a recovery and enhancement of the swelling capacity. CS-AgNPs PAM-GO presented the best swelling capacity in the set of all the hydrogels tested.

Regarding dye removal application, all graphene oxide-based hydrogels in general have a higher adsorption capacity of dyes from their surroundings comparing to PAM, the charges of the dye favored a stronger interaction with PAM-GO hydrogel. Among them PAM-GO and PAM-GO containing PEI-NaBH4 AgNPs are the most efficient adsorbents for R6G.

It was not possible to obtain a hybrid hydrogel, containing GO and AgNPs, with both adsorption and swelling capacities enhanced. Nevertheless, the characteristics obtained make of PAM-GO and PEI-NaBH4 AgNPs hydrogels a potential wastewater dye removal material.

5. References [1] Z. Zhao, X. Wang, J. Qiu, J. Lin, and D. Xu, “Three-

Dimensional Graphene-Based Hydrogel / Aerogel Materials,” Rev. Adv. Mater. Sci, vol. 36, no. 2, pp. 137–151, 2014.

[2] C. He, Z. Shi, L. Ma, C. Cheng, C. Nie, M. Zhou, and C. Zhao, “Graphene oxide based heparin-mimicking and hemocompatible polymeric hydrogels for versatile biomedical applications,” J. Mater. Chem. B Mater. Biol. Med., vol. 3, pp. 592–602, 2014.

[3] I. Tokarev and S. Minko, “Stimuli-responsive hydrogel thin films,” Soft Matter, vol. 5, no. 3, p. 511, 2009.

[4] B. Adhikari, A. Biswas, and A. Banerjee, “Graphene oxide-based hydrogels to make metal nanoparticle-containing reduced graphene oxide-based functional hybrid hydrogels,” ACS Appl. Mater. Interfaces, vol. 4, no.

10

10, pp. 5472–5482, 2012. [5] Z. Fan, B. Liu, J. Wang, S. Zhang, Q. Lin, P. Gong, L. Ma,

and S. Yang, “A novel wound dressing based on Ag/graphene polymer hydrogel: Effectively kill bacteria and accelerate wound healing,” Adv. Funct. Mater., vol. 24, no. 25, pp. 3933–3943, 2014.

[6] Y. Chen, L. Chen, H. Bai, and L. Li, “Graphene oxide–chitosan composite hydrogels as broad-spectrum adsorbents for water purification,” pp. 1992–2001, 2013.

[7] J. N. Tiwari, K. Mahesh, N. H. Le, K. C. Kemp, R. Timilsina, R. N. Tiwari, and K. S. Kim, “Reduced graphene oxide-based hydrogels for the efficient capture of dye pollutants from aqueous solutions,” Carbon N. Y., vol. 56, pp. 173–182, 2013.

[8] S. M. Andrade, C. J. Bueno-Alejo, V. V. Serra, J. M. M. Rodrigues, M. G. P. M. S. Neves, A. S. Viana, and S. M. B. Costa, “Anchoring of Gold Nanoparticles on Graphene Oxide and Noncovalent Interactions with Porphyrinoids,” ChemNanoMat, vol. 1, no. 7, pp. 502–510, 2015.

[9] H. Guo, T. Jiao, Q. Zhang, W. Guo, Q. Peng, and X. Yan, “Preparation of Graphene Oxide-Based Hydrogels as Efficient Dye Adsorbents for Wastewater Treatment.,” Nanoscale Res. Lett., vol. 10, no. 1, p. 931, 2015.

[10] X. Huang, X. Qi, F. Boey, and H. Zhang, “Graphene-based composites,” Chem. Soc. Rev., vol. 41, no. 2, pp. 666–686, 2012.

[11] S. Bhattacharya and S. K. Samanta, “Soft-Nanocomposites of Nanoparticles and Nanocarbons with Supramolecular and Polymer Gels and Their Applications,” Chem. Rev., vol. 116, no. 19, pp. 11967–12028, 2016.

[12] X.-F. Zhang, Z.-G. Liu, W. Shen, and S. Gurunathan, “Silver Nanoparticles: Synthesis, Characterization, Properties, Applications, and Therapeutic Approaches,” Int. J. Mol. Sci., vol. 17, no. 9, p. 1534, 2016.

[13] V. V Mody, R. Siwale, A. Singh, and H. R. Mody, “Introduction to metallic nanoparticles,” J. Pharm. bioallied Sci., vol. 2, no. 4, pp. 282–289, 2010.

[14] J. A. Spadaro, T. J. Berger, S. D. Barranco, S. E. Chapin, and R. O. Becker, “Antibacterial effects of silver electrodes with weak direct current.,” Antimicrob. Agents Chemother., vol. 6, no. 5, pp. 637–42, 1974.

[15] J. S. Kim, E. Kuk, K. N. Yu, J. H. Kim, S. J. Park, H. J. Lee, S. H. Kim, Y. K. Park, Y. H. Park, C. Y. Hwang, Y. K. Kim, Y. S. Lee, D. H. Jeong, and M. H. Cho, “Antimicrobial effects of silver nanoparticles,” Nanomedicine Nanotechnology, Biol. Med., vol. 3, no. 1, pp. 95–101, 2007.

[16] K. Kalishwaralal, S. BarathManiKanth, S. R. K. Pandian, V. Deepak, and S. Gurunathan, “Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis,” Colloids Surfaces B Biointerfaces, vol. 79, no. 2, pp. 340–344, 2010.

[17] H. Mittal, A. Maity, and S. S. Ray, “Gum karaya based hydrogel nanocomposites for the effective removal of cationic dyes from aqueous solutions,” Appl. Surf. Sci., vol. 364, pp. 917–930, 2016.

[18] E. Bozkurt, M. Acar, Y. Onganer, and K. Meral, “Rhodamine 101–graphene oxide composites in aqueous solution: the fluorescence quenching process of rhodamine 101,” Phys. Chem. Chem. Phys., vol. 16, pp. 18276–18281, 2014.

[19] R. Arasteh, M. Masoumi, A. M. Rashidi, L. Moradi, V. Samimi, and S. T. Mostafavi, “Adsorption of 2-nitrophenol by multi-wall carbon nanotubes from aqueous solutions,” Appl. Surf. Sci., vol. 256, no. 14, pp. 4447–4455, 2010.

[20] M. Liu, H. Zhao, X. Quan, S. Chen, and X. Fan, “Distance-independent quenching of quantum dots by nanoscale-graphene in self-assembled sandwich immunoassay.,” Chem. Commun. (Camb)., vol. 46, no. 42, pp. 7909–7911, 2010.

[21] M. Liu, Q. Zhang, H. Zhao, S. Chen, H. Yu, Y. Zhang, and X. Quan, “Controllable oxidative DNA cleavage-dependent regulation of graphene/DNA interaction.,” Chem. Commun. (Camb)., vol. 47, no. 14, pp. 4084–6,

2011. [22] K. Krishnamoorthy, M. Veerapandian, L. Zhang, K. Yun,

and S. Kim, “Antibacterial Efficiency of Graphene Nanosheets against Pathogenic Bacteria via Lipid Peroxidation,” J. Phys. Chem. B, vol. 116, pp. 17280–17287, 2012.

[23] D. Wei, W. Sun, W. Qian, Y. Ye, and X. Ma, “The synthesis of chitosan-based silver nanoparticles and their antibacterial activity,” Carbohydr. Res., vol. 344, no. 17, pp. 2375–2382, 2009.

[24] H. J. Lee, S. G. Lee, E. J. Oh, H. Y. Chung, S. I. Han, E. J. Kim, S. Y. Seo, H. Do Ghim, J. H. Yeum, and J. H. Choi, “Antimicrobial polyethyleneimine-silver nanoparticles in a stable colloidal dispersion,” Colloids Surfaces B Biointerfaces, vol. 88, no. 1, pp. 505–511, 2011.

[25] A. Zielińska, E. Skwarek, A. Zaleska, M. Gazda, and J. Hupka, “Preparation of silver nanoparticles with controlled particle size,” Procedia Chem., vol. 1, no. 2, pp. 1560–1566, 2009.

[26] S. L. Burrs, D. C. Vanegas, M. Bhargava, N. Mechulan, P. Hendershot, H. Yamaguchi, C. Gomes, and E. S. McLamore, “A comparative study of graphene-hydrogel hybrid bionanocomposites for biosensing.,” Analyst, vol. 140, no. 5, pp. 1466–76, 2015.

[27] H. Hu, J. H. Xin, and H. Hu, “PAM/graphene/Ag ternary hydrogel: Synthesis, characterization and catalytic application,” J. Mater. Chem. A, vol. 2, no. 29, pp. 11319–11333, 2014.

[28] B. Adhikari, A. Biswas, and A. Banerjee, “Graphene oxide-based supramolecular hydrogels for making nanohybrid systems with Au nanoparticles,” Langmuir, vol. 28, no. 2, pp. 1460–1469, 2012.

[29] N. T. Nguyen and J. H. Liu, “A green method for in situ synthesis of poly(vinyl alcohol)/chitosan hydrogel thin films with entrapped silver nanoparticles,” J. Taiwan Inst. Chem. Eng., vol. 45, no. 5, pp. 2827–2833, 2014.

[30] Y. Murali Mohan, K. Vimala, V. Thomas, K. Varaprasad, B. Sreedhar, S. K. Bajpai, and K. Mohana Raju, “Controlling of silver nanoparticles structure by hydrogel networks,” J. Colloid Interface Sci., vol. 342, no. 1, pp. 73–82, 2010.

[31] W. Cui, J. Ji, Y.-F. Cai, H. Li, and R. Ran, “Robust, anti-fatigue, and self-healing graphene oxide/hydrophobically associated composite hydrogels and their use as recyclable adsorbents for dye wastewater treatment,” J. Mater. Chem. A, vol. 3, no. 33, pp. 17445–17458, 2015.

[32] V. Dhanapal and K. Subramanian, “Modified chitosan for the collection of reactive blue 4, arsenic and mercury from aqueous media,” Carbohydr. Polym., vol. 117, pp. 123–132, 2015.

[33] C. T. Chien, S. S. Li, W. J. Lai, Y. C. Yeh, H. A. Chen, I. S. Chen, L. C. Chen, K. H. Chen, T. Nemoto, S. Isoda, M. Chen, T. Fujita, G. Eda, H. Yamaguchi, M. Chhowalla, and C. W. Chen, “Tunable photoluminescence from graphene oxide,” Angew. Chemie - Int. Ed., vol. 51, no. 27, pp. 6662–6666, 2012.

[34] D. C. Marcano, D. V Kosynkin, J. M. Berlin, a Sinitskii, Z. Z. Sun, a Slesarev, L. B. Alemany, W. Lu, and J. M. Tour, “Improved Synthesis of Graphene Oxide,” ACS Nano, vol. 4, no. 8, pp. 4806–4814, 2010.

[35] S. Yang, X. Feng, S. Ivanovici, and K. Müllen, “Fabrication of graphene-encapsulated oxide nanoparticles: Towards high-performance anode materials for lithium storage,” Angew. Chemie - Int. Ed., vol. 49, no. 45, pp. 8408–8411, 2010.

[36] Y. J. Zhang, “Investigation of Gold and Silver Nanoparticles on Absorption Heating and Scattering Imaging,” Plasmonics, vol. 6, no. 2, pp. 393–397, 2011.

[37] D. Y. Wang, D. W. Wang, H. A. Chen, T. R. Chen, S. S. Li, Y. C. Yeh, T. R. Kuo, J. H. Liao, Y. C. Chang, W. T. Chen, S. H. Wu, C. C. Hu, C. W. Chen, and C. C. Chen, “Photoluminescence quenching of graphene oxide by metal ions in aqueous media,” Carbon N. Y., vol. 82, no. C, pp. 24–30, 2015.

[38] H. Bai, C. Li, X. Wang, and G. Shi, “On the gelation of

11

graphene oxide,” J. Phys. Chem. C, vol. 115, no. 13, pp. 5545–5551, 2011.

[39] J. Fan, Z. Shi, M. Lian, H. Li, and J. Yin, “Mechanically strong graphene oxide/sodium alginate/polyacrylamide nanocomposite hydrogel with improved dye adsorption capacity,” J. Mater. Chem. A, vol. 1, no. 25, p. 7433, 2013.

[40] ISS Inc., “ISS Data Tables | Lifetime Data of Selected Fluorophores,” 2013. [Online]. Available: http://www.iss.com/resources/reference/data_tables/LifetimeDataFluorophores.html. [Accessed: 24-Oct-2016].

[41] N. Zhang, R. Li, L. Zhang, H. Chen, W. Wang, Y. Liu, T. Wu, X. Wang, W. Wang, Y. Li, Y. Zhao, and J. Gao, “Actuator materials based on graphene oxide/polyacrylamide composite hydrogels prepared by in situ polymerization,” Soft Matter, vol. 7, no. October, pp. 7231–7239, 2011.