In Situ Synthesis of Silver Nanoparticles within Hydrogel-conjugated

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In Situ Synthesis of Silver Nanoparticles within Hydrogel-conjugated Membrane for Enhanced Anti-Bacterial Properties Deepika Somayajula, Ayushi Agarwal, Ajay Sharma, Ashley Pall, Saurav Datta, and Gargi Ghosh ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00471 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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ACS Applied Bio Materials

Membranes with immobilized AgNPs Characterization and performance assessment

Membranes with encapsulated AgNPs

Modification of commercially available membranes

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In Situ Synthesis of Silver Nanoparticles within Hydrogelconjugated Membrane for Enhanced Anti-Bacterial Properties

Deepika Somayajula1, Ayushi Agarwal2, Ajay K. Sharma2, Ashley E. Pall3, Saurav Datta2*, Gargi Ghosh1*

1: Department of Mechanical Engineering, University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, MI-48128 2: Department of Biotechnology, Indian Institute of Technology Roorkee, India 3: Department of Natural Sciences, University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, MI-48128, USA

*Corresponding Author: email address: [email protected], Phone: 1-313-593-5013, Fax: 1-313593-3851 *Corresponding Author: email address: [email protected], Phone: 91-1332-284795

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Abstract Biofouling negatively impacts water treatment performance of membranes by reducing water permeability, increasing energy consumption, and shortening the lifetime of the membranes. In this study, we integrated the bactericidal property of silver nanoparticles (AgNPs) with hydrophilicity of hydrogels to modify membranes that not only reduce adhesion, but also deactivate the adhered bacteria. Two approaches for AgNP synthesis were adopted – in situ synthesis and encapsulation in single step, and immobilization in multi step. Formation of AgNPs was confirmed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) studies. Compared to the pristine membrane, AgNP/hydrogel modified membranes displayed no adverse effect in water flux under gravitational flow condition. The AgNP/hydrogel modified membranes also exhibited better antibacterial properties (inhibition of adhesion and growth of E.coli) as demonstrated by the bacterial growth, inhibition zone and co-culture (with the membranes) studies. The improvements could be attributed to the synergistic effect of hydrophilic hydrogel networks and the presence of bactericidal AgNPs. In addition, comparison of the anti-bacterial studies revealed the superiority of the encapsulated membrane over the immobilized membrane. This could be attributed to the efficient release of the former over the latter. To the best of our knowledge, this is the first study that demonstrates the enhancement of anti-bacterial properties of membrane via in situ synthesis and encapsulation of AgNPs within hydrogel matrices. Keywords: Silver nanoparticles, Membranes, Hydrogels, Anti-bacterial, Water treatment



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Introduction Access to clean water is not only critical for human health and survival, but also for socioeconomic development, pharmaceuticals and food production, and the environment at large. Inadequate and inappropriate management of water, resulting in millions of people drinking contaminated or chemically polluted water, has been recognized as one of the most challenging global problems. According to the World Health Organization, globally, at least two billion people drink contaminated water and by 2025 half of world’s population is expected to live in water-stressed areas (http://www.who.int/news-room/fact-sheets/detail/drinking-water). High separation performance, low cost, and ease of operation and scalability have led to wide application of membrane based water filtration systems in water purification and wastewater treatment1, 2. However, fouling caused by the accumulation and growth of foulants on membrane matrix limits the applications of membranes. Of all types of fouling, biofouling, due to the deposition of microbes, and subsequent growth and biofilm formation on membrane matrix, is the most complicated challenge in membrane separation processes3-5. Biofouling reduces the transport of purified water (quantity) across the membrane, changes the rejection characteristics (quality) and leads to a shorter lifetime (cost) of the membrane3, 4, 6. Due to their excellent anti-microbial properties, silver nanoparticles (AgNPs) and silverbased compounds are widely used in wound/burn dressings7,

8

and medical implants and

instruments9, 10. The anti-bacterial properties of silver have been attributed to the ability of silver ions to interact with the thiol groups present in the cysteine residues of the bacterial proteins11. The formation of S-Ag bonds results in the damage of bacterial proteins, interruption of electron transport chain, and DNA dimerization11-13. In addition, studies also reported damage of bacterial


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cell wall upon exposure of bacteria to AgNPs11. The consequent cell death has been attributed to increased cell permeability leading to osmotic collapse and release of intracellular material11. Research efforts have been directed towards incorporating AgNPs into membrane matrix including physical blending or surface coating to enhance the anti-biofouling property of the polymeric membranes11,

14-17

. However, the high surface energy of AgNPs results in their

aggregation in water, and thereby, impairs their anti-bacterial properties unless they are physically blended or surface coated. Immobilization of AgNPs on membrane surface could yield better homogeneity and impart stability of these nanoparticles. Examples of immobilization of AgNPs on various membrane matrix for improving the antifouling properties include: (i) polyamide thin-film layer18,

19

, (ii) sulfonated polyethersulfone membrane20, (iii) multiwalled

carbon nanotube coated hollow fiber membrane17 [24], (iv) polyethyleneimine coated ultrafiltration membrane21, (v) polyamide thin-film composite membrane22, and (vi) graphene oxide conjugated polyvinylidene fluoride membrane23. The findings from these studies highlight the importance of presence of AgNPs at the feed interface of the membrane to permit direct contact between silver and bacterial cells for improved antimicrobial performance. While modification of membranes with AgNPs permit inhibition of bacterial growth and eventually lead to death, accumulation of dead bacteria on the membrane surfaces promotes membrane fouling resulting in decline in membrane performance (permeability and lifetime)6. One of the commonly used methods for reducing membrane fouling is the application of hydrophilic surface coatings on to the membranes without significantly compromising water flux24-27. Hydrogels, three dimensional polymeric network architectures capable of absorbing large amount of water, have gained significant attention as coatings by virtue of being versatile, tunable, functionalizable, and inherently antifouling owing to the hydrophilicity. Hydrogel 4 ACS Paragon Plus Environment


membranes, primarily reported in literature, are either free-standing films28-30 or films attached physically or chemically with/at the membrane interfaces. Different methods had been adopted to create hydrogel films on porous membrane surfaces. These include immersion of double bond grafted PES membranes into hydrogel precursor solution followed by free-radical crosslinking31 and casting the hydrogel precursor solution consisting of acrylic acid/2-hydroxyethyl methacrylate onto the membrane surface followed by photo-initiated graft polymerization32. Besides, techniques, such as, UV-curing hydrogel precursor solutions spread over membranes33 and utilizing layer-by-layer “click” chemistry for fabrication of ultrathin hydrogel films,34 had also been used. Another approach was coating membrane surface with ene-functionalized dopamine followed by layer-by-layer deposition of thiol-functionalized poly (oligo(ethylene glycol)mercaptosuccinate) and ene-functionalized poly (sulfobetaine methacrylate-co-acrylate acid)35. Here, we integrated the benefits of the bactericidal property of AgNPs with hydrophilicity of polymeric hydrogel within the same matrix and report development of a novel AgNP-hydrogel conjugated membrane with improved anti-bacterial characteristics. The goal of the study was to impart bactericidal properties to commercially available nylon membranes and investigate the impact of modification on membrane performance. Towards this goal, a novel approach was adopted for in situ generation of AgNPs within hydrogel matrices. Nylon membranes were incubated with a polymeric precursor solution consisting of a blend of 4-arm poly (ethyleneglycol) acrylate (PEG acrylate), poly (acrylic acid) (PAA), and photo-initiator in the presence of silver nitrate solution. The free radicals generated during photo-polymerization of PEG acrylate and PAA triggered simultaneous formation and encapsulation of AgNPs within the hydrogel networks on membrane surfaces. Changes in physical properties of the membranes due


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to the modification was examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM), while changes in chemical properties of the surfaces due to the modification was investigated by energy dispersive x-ray spectroscopy (EDX) and x-ray dispersion (XRD) characterization and compared with membranes modified via multi-step immobilization method. In the immobilization method, initially a hydrogel layer (blend of PEG acrylate/PAA) was formed on membrane surface via exposure to UV radiation. The hydrogel-conjugated membranes were then exposed to silver nitrate and sodium borohydride successively to immobilize AgNPs on the gel surfaces. The performances of the surface modified membranes were systematically assessed in terms of water flux and anti-bacterial properties using E. coli as the model organism. Dissolution and release of AgNPs (Ag+) are necessary for the anti-bacterial properties of the membranes; however, persistent release and consequent depletion of silver reduce the efficacy of the modified membranes. To establish the long-term operational stability of the membranes, anti-bacterial properties were investigated for extended period of time.

1. Materials and Methods 1.1 Materials All compounds were used as received. 4Arm-PEG-Acrylate (MW10K) (PEG acrylate) was obtained from Layson Bio Inc. Poly(acrylic acid) (PAA) (MW45,000), photo-initiator (PI) 2Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (98%), sodium hydroxide, silver nitrate, sodium borohydride powder (≥98.0%), and ethylene glycol (Reagent Plus, ≥99%) were purchased from Sigma-Aldrich (St. Louis, MO). Hydrochloric acid (HCl) was purchased from Fisher Scientific (Pittsburgh, PA). Nylon membranes (0.45 µm pore size, 47 mm disc diameter) were procured from Millipore. The membrane experiments were performed in a solvent resistant



stirred ultrafiltration cell (Millipore, Model XFUF04701) under gravitational flow condition with dead-end filtration mode. 1.2 Membrane modification In situ generation and encapsulation of AgNPs within hydrogel matrix: Nylon membrane was selected, because of its hydrophilicity and existence of amine groups, which could be reacted further for modification. In a water treatment process, prior to ultrafiltration (UF), a microfiltration (MF) membrane is typically used for bacterial cell retention. This study aims to develop a MF membrane with improved anti-bacterial properties. To achieve that, a commercially available 0.45 μm MF membrane was modified with AgNPs and polymeric hydrogel, such that the solvent transport characteristics remain unchanged. For modification of membranes via encapsulation of AgNPs within hydrogel matrix, nylon membranes were first acid treated to expose the amine groups. Acid or base hydrolysis of amide bonds is a widely used and well established technique to expose amine groups of nylon membrane36-38. This was conducted by incubating nylon membranes in 5 mL of 1 N HCl and moderately shaking for approximately 30 min at room temperature (25 oC). The acid treated nylon membranes were then immersed in 5 mL precursor solution consisting of PEG acrylate (0.4 % wt/vol), PI (1 % wt/vol), PAA (0.025 % wt/vol) and silver nitrate (2.0 % wt/vol) and incubated for 5 h at 60 oC. Following incubation, the membranes were removed from the solution and UV cured for 5 min using CL-1000 ultraviolet crosslinker. Exposure to UV resulted in the formation of AgNPs39. Immobilization of AgNPs within hydrogel matrix: For immobilization of AgNPs, acid treated nylon membranes were incubated in 5 mL polymer precursor solution consisting of PEG acrylate (0.4 % wt/vol), PI (1 % wt/vol), and PAA (0.025 % wt/vol) for 5 h at 60 oC. Post incubation, the membranes were removed from the precursor solution and exposed to UV as described above.


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Using the stirred filtration cell, 10 mL of NaOH (2 % wt/vol) was permeated through the membranes under gravitational flow condition. The membranes were then washed with deionized water until the pH of the supernatant is balanced to 7.0. To immobilize silver ions, the membranes were then immersed in 8 mL of silver nitrate solution (2.0 % wt/vol) for 4 h under stirred condition at room temperature. Finally, 5 mL of sodium borohydride solution (10 % wt/vol) was added to convert silver ions to AgNPs. After 4 h of reactions, the membranes were washed with deionized water to remove the unreacted reagents and silver ions. 2.3 Membrane characterization Morphological changes in the modified membranes were observed using a Field EmissionScanning Electron Microscope (FE-SEM, FEI Quanta 200 F) at an accelerating voltage of 15−20 keV. Dried samples of the unmodified and modified membranes were sputter coated with gold in BAL-TEC SCD 005 gold sputtering unit and mounted on FE-SEM stage for analysis. The sample specimens were examined at 10K-25K magnifications. Changes in chemical composition due to modification were examined by an Energy Dispersive X-ray Spectroscopy (EDX) unit attached to the FE-SEM unit. Compositional changes due to modification were also evaluated using X-ray Diffraction (XRD) (Rigaku Miniflex). XRD patterns of the membranes were recorded using Cu Kα radiation and 2θ angles from 10o to 90o. The average particle size of AgNPs was calculated using Debye-Scherrer equation40 D=Kλ/βCosθ

(1)

where D is crystalline size of AgNPs, λ the wavelength of X-ray source (0.154) used in XRD, β is full width at half maximum of diffraction peak, K Scherrer constant with value of 0.9 to 1, and θ is Bragg angle.



The influence of AgNP-modification on the topology of the membrane surfaces was studied via Atomic Force Microscopy (AFM) (Hitachi) analysis (tapping mode). The surface roughness of the membranes was based on AFM topography images of 5.0 µm X 5.0 µm scan area. The release of silver ions from the membranes was analyzed using an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) (Perkin Elmer Elan DRD). The membranes were incubated in 20 mL deionized water and placed on an orbital shaker (100 rpm) for the desired period of time. Following incubation, the samples were collected, treated with 1% nitric acid, and the silver content was analyzed. 2.4 Water flux assessment Water flux assessment experiments were conducted using the stirred filtration cell under gravitational flow condition, i.e. in absence of any external pressure except the water head. Volume of feed water was varied to obtain different water head, and hence, the applied transmembrane pressure. Flux experiments were performed with all three membranes – pristine, immobilized and encapsulated. Cumulative volume of pure water (deionized water) permeated through the membranes at a certain time was measured. Then, permeate flux was calculated by the following expression: J=V/(A×𝑡), where J is the permeate flux, V is the cumulative volume of permeate collected at permeation time of t and A is the effective membrane surface area (15 cm2). 2.5 Anti-bacterial property assessment Anti-bacterial property of the pristine and silver modified membranes was investigated with E. coli, the most widely studied Gram-negative bacteria, as the model organism. Fresh cultures of E. coli (DH5-Alpha) were prepared by transferring a loop-full of stock culture into a 300 mL Luria-Bertani (LB) nutrient broth (Fisher BioReagents, LB Broth, Miller) and grown overnight 9 ACS Paragon Plus Environment

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(16-18 h) at 37 0C in a shaker (~220 rpm). Density of cells was quantified by measuring the optical density at 600 nm (OD600) using a UV-VIS Spectrophotometer. To evaluate the antibacterial activity of the membranes, LB agar plates were streaked with loop-full of E. coli culture and the membranes were placed on the plates with the active side facing downwards to ensure direct interaction with the agar and the bacteria. After incubation at 37 0C for 24 h, the inhibition zones around the membranes were monitored. To assess the ability of the modified membranes to prevent bacterial adhesion, 5 mL of E.coli solution (10% of bacterial solution in LB broth diluted in 90% water) was permeated through the pristine as well as the silver modified membranes using the stirred filtration cell. The OD600 of the feed and permeate solutions were measured to calculate rejection of bacteria by different membranes. Following permeation of bacterial solutions, membranes were immersed in 50 mL of media and incubated at 37 0C overnight. The OD600 of the media was measured on the following day to estimate the viability of the adhered cells. 2.6 Statistical Analysis All experiments were repeated at least three times unless mentioned otherwise. The data represents the mean ± standard error mean of three independent experiments. Two data sets considered significantly different at a p-value < 0.05 (analyzed via ANOVA factorial method).

2. Results and Discussion 2.1 Membrane modification For in situ AgNP generation, the acid treated nylon membrane was soaked into the polymer precursor solution containing PEG acrylate, PAA and the PI along with silver nitrate. Incomplete Michael addition reaction between the amine groups of nylon membrane and the acrylate groups of PEG acrylate resulted in the conjugation of the latter to the membrane surface. The residual 10 ACS Paragon Plus Environment


acrylate groups were crosslinked in presence of the UV radiation. This resulted in the formation of PEG acrylate/PAA hydrogel network on the membrane surface (Fig 1), which endowed the membranes with anti-bacterial properties. In addition, the free radicals

Figure 1. Schematic demonstrating the modification of nylon membranes to endow them with anti-bacterial properties. The reaction scheme involves incomplete Michael addition reaction between the amine groups on membrane surface and the acrylate groups in the hydrogel precursor solution. Further crosslinking upon exposure to UV permits the formation of 3D hydrogel matrices on the membrane surfaces. Silver modification was carried out either by incubating the hydrogel coated membranes with AgNO3 solution followed by reduction in the presence of sodium borohydride or by incorporation of AgNO3 within precursor solution.


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(cleavage of photo-initiator) formed upon exposure to UV radiation41 was exploited for in situ generation and stabilization of AgNPs within the hydrogel networks. AgNP formation involved two parallel processes: photo-reduction of silver nitrate to form AgNPs and photosensitization, which involved the reduction of silver ions by photochemically generated free radicals. These same radicals were also involved in crosslinking the acrylate groups to create the hydrogel network. The transformation of the pristine white colored membranes into black/brown following photo-polymerization indicated the growth of AgNPs within the gel on the membrane surface (Fig 2).

i

ii

iii

iv

Figure 2. Digital images of (i) pristine, (ii) hydrogel coated, (iii) membranes modified via in situ generation and encapsulation of AgNPs and (iv) multi-step method of AgNP immobilization. AgNP immobilization/encapsulation resulted in a change in color of the membrane surface.

Entrapment of PAA within PEG hydrogel network permitted introduction of negative charges (anionic carboxylic acid groups) within the gels. For the multi-step method of immobilization of silver ions, following the formation of hydrogel networks, the membranes were immersed in silver nitrate solution to promote electrostatic interaction between the silver ions and the carboxylic acid groups of PAA. Finally, reduction of the silver ions with sodium borohydride resulted in the formation of AgNPs on the membrane surface as manifested from the transformation of the white colored pristine or hydrogel coated membrane to black/brown upon silver immobilization (Fig 2). 12 ACS Paragon Plus Environment


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2.2 Membrane characterization Figs. 3A show the SEM images of surface of the pristine as well as the modified nylon membranes. Distinct morphological difference between 3A(i) and 3A(ii)/(iii) was observed due A

B

C

i

ii

i

ii

i

ii

iii

iii

Figure 3. A) SEM images and B) corresponding EDX spectra of (i) pristine, (ii) AgNP immobilized, and (iii) AgNP in situ generated membranes. (C) Mapping of silver distribution on membrane surfaces modified by (i) immobilization and (ii) encapsulation of AgNPs.

to the partial coverage of the membrane surface with PEG acrylate/PAA hydrogel network. EDX was used to investigate the deposition of silver within hydrogel network on the membrane surface (Figs. 3B and C). Compared to the pristine membrane, elemental analysis of the modified 13 ACS Paragon Plus Environment

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membranes revealed the presence of characteristic peaks of silver, confirming the incorporation (via immobilization or in situ generation and encapsulation) of silver within the membrane surface (Figs 3B (ii)/(iii)). The weight percentage of incorporated silver was found to be 10 % and 8.5 % for the immobilized and encapsulated membranes, respectively. Mapping (Figs. 3C (i) and (ii)) revealed uniform distribution of elemental silver throughout the surface area of the modified membranes, thereby establishing the efficacy of both of the modification techniques. XRD spectra of nylon membranes coated with PEG acrylate/PAA hydrogel and silver modified membranes are shown in Fig 4. The diffraction peaks at 2θ values of 38.28, 44.7, 64.84, and 77.82 corresponding to (111), (200), (220), and (311) reflection planes confirmed the formation of AgNPs. These diffraction peaks are consistent with the Joint Committee on Powder Diffraction Standards (JCPDS) silver file No. 04-0783. The average crystallite size calculated using Debye-Scherrer equation with the width of the (111) peak was found to be 9 and 13 nm for immobilized and in situ generated and encapsulated silver nanoparticles, respectively. No peaks (broad pattern) implying the amorphous state of synthesized AgNPs were observed in either of the modification methods.



3500

Intensity(a.u)

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3000

Hydrogel coating

2500

Immobilized silver

2000

Encapsulated silver

1500 1000 500 0

10

20

30

40

50

60

70

80

90

2θ (degree)

Figure 4. Comparison of XRD pattern of nylon membranes conjugated with hydrogel networks with the membranes modified by incorporation of AgNPs within the hydrogel matrices. Similar silver patterns were observed irrespective of modification methods.

AFM analysis was performed to determine the impact of incorporation of hydrogel and silver nanoparticles on the surface roughness of the modified membranes. Fig 5 represents the three dimensional AFM images of pristine, hydrogel coated and two AgNP modified membranes. In the AFM images of the pristine, hydrogel coated as well as the modified membranes, nodule valley-like structures could be observed. Surface roughness parameters (Sa-arithmetic mean height and Sq/RMS-Root mean square height) were evaluated from the AFM images (reported in the inset of the images). Hydrogel grafting and silver incorporation increased the roughness of the membranes as compared to the pristine membrane. Further, roughness of AgNP immobilized membranes was found to be lower than the AgNP encapsulated membranes. Presumably, treatment of hydrogel conjugated membranes with silver nitrate and sodium borohydride solutions resulted in the collapse of some PEG/PAA chains during the conventional method.


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A

B

Sa:

94 nm

Sq/RMS:

121 nm

Sa:

114 nm

Sq/RMS:

156 nm

C

Sa:

161 nm

Sa:

186 nm

Sq/RMS:

208 nm

Sq/RMS:

236 nm

Figure 5. AFM topographical images of A) pristine membrane, B) hydrogel-coated membrane, C) immobilized AgNP-modified membrane, and D) in situ generated AgNP-modified membrane. The roughness parameters are shown in the inset.

2.3 Membrane performance assessment The pure water flux values of the pristine as well as the silver modified membranes are presented in Fig 6. As can be observed, surface modification of the membranes did not have any adverse effect on water flux (p-values > 0.05) for a fixed transmembrane pressure drop. As an example, for hydrostatic pressure of 0.005 bar, the pure water flux obtained for pristine membranes, membranes with immobilized AgNPs, and membranes with encapsulated AgNPs were 22.4 ± 1.0 L/m2/h, 21.6 ± 0.8 L/m2/h, and 24.4 ± 0.4 L/m2/h, respectively. Consequently, no significant

D 16 ACS Paragon Plus Environment


30 Pristine 25

Flux (L/m 2/h)

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y = 2823.6x + 8.58

Immobilized

Encapsulated

y = 3221.2x + 5.04

y = 3262x + 7.6

20

15

10

5

0 0

0.001

0.002

0.003

0.004

0.005

0.006

Transmembrane pressure drop (bar)

Figure 6. Comparison of pure water flux vs. transmembrane pressure drop data between the pristine and the AgNP modified membranes (immobilized and encapsulated) under gravitational flow condition. Error bars S.E.M. (N=3).

difference between the slopes of the flux vs transmembrane pressure drop data for the pristine (2824 L/m2/h/bar), immobilized (3221 L/m2/h/bar) and encapsulated (3262 L/m2/h/bar) membranes was observed. The slope of the flux vs. transmembrane pressure drop data indicates the ability of the membrane to transport water. Therefore, the data reveals that the water transport characteristics of the membranes after modifications remained unaltered. Preventing deterioration of water transport properties through membrane during modification is a critical factor, because water transport dictates the productivity of the process. It is significantly influenced by the method of modification. McVerry et al.42 reported a low-fouling modified 17 ACS Paragon Plus Environment

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polysulfone ultrafiltration membrane that exhibited similar flux and rejection characteristics, but improved anti-fouling properties as compare to the unmodified polysulfone membrane. On the other hand, Zhang et al.43 developed a modified polyimide membrane that resulted into decrease in flux due to modification. In our case, although incorporation of polymers increased the resistance to water transport through the membrane, the hydrophilicity of the polymers seemed to nullify that effect.

2.4 Anti-bacterial properties of the membrane surface To evaluate the anti-bacterial property of the membranes modified via in situ generation of AgNPs, bacterial feed of E. coli was permeated through the membranes. The percent rejection of bacteria by the membranes was calculated based on the difference in OD600 of the feed and the permeate solutions. No statistical variation (p-value > 0.05) in the bacterial rejection (> 90%) by all four membranes (pristine, hydrogel coated, immobilized AgNPs and encapsulated AgNPs) was observed. Following permeation of bacterial solutions, the membranes were incubated in bacterial growth media overnight. The goal was to assess the ability of these membranes to reduce the adhesion of bacterial cells and the growth of bacterial colony. OD600 of the media was measured to assess the bacterial growth in the culture media for all four membranes. Then, bacterial cell growth for all four cases was compared on the basis of growth for the pristine membrane and represented in Fig 7. Compare to the pristine membranes, incubation of hydrogel conjugated membranes resulted in a significant decrease in bacterial cell growth in the media (75.0 % ± 10.0 % for hydrogel conjugated membranes). This indicates that PEG acrylate/PAA hydrogels provided an anti-biofouling support by reducing the adhesion of bacterial cells on the surface. La 18 ACS Paragon Plus Environment


et al.44 also reported formation of an ammonium salt-containing PEGDA hydrogel (with no silver) and demonstrated the antimicrobial property with the help of E. coli growth study. The growth of E. coli was further reduced upon incubation of culture media with silver modified membranes (50.1 % ± 4.5 % for immobilized and 1.6 % ± 1.0 % for encapsulated) (p-value < 0.05). The reduced bacterial growth could be either due to (i) fewer live bacteria or (ii) irreversible adsorption of bacteria within the membrane. However, the possibility of irreversible adsorption of whole population of bacteria could be eliminated, because the media, in presence of the pristine, hydrogel-coated and immobilized membranes, demonstrated significant bacterial growth (although at varying extents). This signifies that the bacterial cells were able to transfer from the membrane to the growth media. The media in presence of the encapsulated membrane demonstrated negligible growth. Therefore, the variation in bacterial growth for the four cases represented in Fig 7 must be due to the presence of different counts of live bacteria within the membranes. The encapsulated membrane contained least count of live bacteria (due to either least adhesion or destruction of the adhered bacterial cells), and therefore, exhibited least growth in the media. These observations indicate that incorporation of AgNPs within the hydrogel networks conjugated to the membrane surfaces not only effectively resisted adhesion of bacteria cells, but also facilitated destruction of the bacteria cells attached to the surfaces. Reduced viability of adsorbed bacteria attributed to low growth of cells in culture media. The efficacy of the membranes modified via encapsulation of AgNPs in exterminating bacterial cells was found to be superior to the membranes modified via immobilization of AgNPs. This could be attributed to the enhanced release/diffusion of silver ions from the encapsulated membranes compared to the immobilized membranes. The silver ion release data for the immobilized and encapsulated membranes are compared at the end of this section to justify the above statement.


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Bacterial cell growth relative to pristine (%)

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120 100

*

*

Hydrogel coated

Immobilized

*

80 60 40 20

0 Pristine

Encapsulated

Membranes

Figure 7. Comparison of bacterial cell growth in presence of pristine, hydrogel coated, immobilized AgNPs containing and encapsulated AgNPs containing membranes. Inhibition of bacterial growth in media is compared on the basis of the pristine membrane. Error bars S.E.M (N=3). * p-value < 0.05.

To assess the long-term stability of AgNPs within the modified membranes, separate set of experiments were conducted with “used” encapsulated and immobilized membranes. “Used” membrane indicates a membrane that was subjected to about 2 L of water permeation and 4 months of storage under deionized water at room temperature. The used membranes (pristine as well as modified membranes) were placed on E. coli streaked LB agar plates and incubated overnight. LB plate streaked with E. coli without any membrane was employed to serve as the control. As illustrated in Figs. 8A, there was no inhibition zone surrounding the pristine membrane indicating the lack of antibacterial effect. Whereas, in the presence of the silver modified membranes, the bacterial colonies did not grow near the membranes and led to the formation of inhibition zone. There are previously reported studies on encapsulated AgNPs on 20 ACS Paragon Plus Environment


polyethersulfone35, polyimide43 and polyamide22 membranes with antibacterial characteristics demonstrated by inhibition zone study similar to that of Figs. 8A. However, the studies were conducted for shorter period of time (immediately - He et al., 2018 and Zhang et al., 2018; after 14 days - Yin et al., 2013), whereas in this case, silver modified membranes were stored in deionized water for 4 months prior to the test. Inhibition of E. coli growth in the presence of these membranes elucidated that the anti-biofouling property of the silver modified membranes due to the bactericidal action of AgNPs were retained even after substantial usage and long term storage. Furthermore, the results also highlighted significantly higher inhibitory effect of the membrane with encapsulated AgNPs. In addition, the “used” membranes were also introduced into E. coli culture, incubated for 8 h at 37 0C and the effect of membrane on E. coli growth was studied. Figs. 8B represent the images of the E. coli culture after 8 h of incubation with different membranes. In case of the pristine (Fig 8B (ii)) as well as the AgNP immobilized (Fig 8B (iii)) membranes, significant growth of bacteria was observed as reflected from the cloudiness of the media. The pristine membrane did not have any bactericidal action, hence as expected, the microorganisms grew without any inhibition. For the AgNP immobilized membrane, although growth was less than that for the pristine membrane, the cloudiness suggests lack of bactericidal action after prolong usage and storage. In contrary, for the AgNP encapsulated membrane (Fig 8B (iv)), growth of E. coli was not at all observed and the culture was as clear as the media itself (Fig 8B (i)). This could be attributed to the preservation of the bactericidal action of the AgNP encapsulated membrane over prolonged period of time. Inhibition of bacterial cell growth, while co-culturing E. coli cells with the AgNP modified membranes, were reported by other research groups as well22, 35, 43. However, the AgNP encapsulated membrane, in this case, demonstrated its effectiveness even after four


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months of long storage period. The intactness of the bactericidal action of the AgNP encapsulated membrane establishes the long-term stability of the same and emphasizes the superiority of it over AgNP immobilized membrane.

A i

iii

ii

B

Figure 8. A) Images of E. coli streaked LB agar plates demonstrating antibacterial properties of (i) pristine membrane, (ii) membrane with immobilized AgNPs, and (iii) membrane with encapsulated AgNPs. B) Images showing bacterial growth in media upon incubation with various membranes for 8 h at 37 0C. (i) Culture media, (ii) pristine membrane and media, (iii) immobilized AgNPs and media, and (iv) encapsulated AgNPs and media.

The efficacy of anti-bacterial properties of the modified membranes was associated with the release of silver ions from the modified membranes. However, rapid release of silver ions limits the long-term application of the modified membranes. In this study, the release characteristic of 22 ACS Paragon Plus Environment


silver ions over a period of time (2 and 7 days) was analyzed via ICP-MS. The release of silver ions was found to be 0.02% ± 0.005% and 1.1% ± 0.5% of the loaded silver ions after 2 days of incubation in deionized water from immobilized and encapsulated membranes, respectively. Also, the release of silver ions was around 0.075% ± 0.02% and 2.5% ± 0.6% of the loaded silver ions after 7 days of incubation for the immobilized and encapsulated membranes, respectively. Taking into account the controlled release rate of silver ions from the membranes, it is expected that the membranes will retain their anti-bacterial properties for longer period of time as demonstrated above. The above data also reveal that the AgNP encapsulated membrane has better release efficiency of silver ions over the AgNP immobilized membrane.

3. Conclusions We report enhancing the anti-bacterial properties of commercially available nylon membrane by synergistically combining the bactericidal property of AgNPs with hydrophilic property of hydrogels. Our approach was based on exploiting the incomplete Michael addition reaction to incorporate hydrogel network of a blend of polyethylene glycol acrylate and polyacrylic acid on nylon membrane surface. One-step modification method involving in situ generation and encapsulation of AgNPs within hydrogel network was compared with the multi-step method of AgNP immobilization. Both of the methods of incorporation of AgNPs within membranes resulted into uniform distribution of nanomaterials as confirmed by various characterization studies. Incorporation of functional components did not adversely affect the permeability of the modified membranes. Both of the modified membranes were able to resist bacterial adhesion and growth as demonstrated with E. coli as the model microorganism. Hydrophilic property of hydrogel prevented the adhesion, whereas bactericidal property of AgNPs prevented the growth


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of bacteria. AgNP encapsulated membrane was observed to be superior to the AgNP immobilized membrane. This could be attributed to the efficient release and diffusion of silver ions from the former one compare to the latter one as demonstrated by the silver release study. The AgNP encapsulated membrane also demonstrated superior long term stability compared to the AgNP immobilized membrane. Overall, the study led to a facile but efficient method of in situ formation and encapsulation of AgNPs within hydrogel-conjugated membrane, which endowed the membrane with improved anti-bacterial properties.

4. Acknowledgements The authors would like to acknowledge Trehan Foundation (Grant Number: G018637) and Ramanujan Fellowship Grant, SERB, India (Grant number: SB/S2/RJN-028/2014) for financial support of this study. The authors would also like to thank Dr. Kalyan Kondapalli for helpful discussion and guidance in the bacterial study and Corey Lambert for helping with ICP-MS analysis.



References 1. Z. Wang, H. Yu, J. Xia, F. Zhang, F. Li, Y. Xia, Y. Li, Novel GO-blended PVDF Ultrafiltration Membranes, Desalination 299 (2012) 50-54. 2. W. Gao, H. Liang, J. Ma, Han, Z-L. Chen, Z.-S. Han, G.-B. Li, Membrane Fouling Control in Ultrafiltration Technology for Drinking Water Production: a Review, Desalination 272 (2011) 1-8. 3. M. Herzberg, M. Elimelech, Biofouling of Reverse Osmosis Membranes: Role of Biofilm-enhanced osmotic pressure, J. Membr. Sci. 295 (2007) 11-20. 4. M. Herzberg, M. Elimelech, Physiology and Genetic Traits of Reverse Osmosis Membrane Biofilms: a case study with Pseudomonas aeruginosa, ISME J. 2 (2008) 180194. 5. J. Dolina, O. Dlask, T. Lederer, L. Dvorak, Mitigation of Membrane Biofouling through Surface Modification with Different forms of Nanosilver, Chem. Eng. J. 275 (2015) 125133. 6. Y. Yang, C. Li, Li-an. Hou, Impact of Dead Cells on Biofouling and Pharmaceutically Active Compounds Retention by NF/RO Membranes. Chem. Eng. J. 337 (2018) 51-59. 7. N.S.V. Capanema, A.A.P Mansur, S.M. Carvalho, L.L. Mansur, C.P. Ramos, A.P. Lage, H.S. Mansur, Physicochemical Properties and Antimicrobial Activity of Biocompatible Carboxymethylcellulose-silver Nanoparticle Hybrids for Wound Dressing and Epidermal Repair. J. Appl. Polym. Sci. 135 (2018), 45812.


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8. J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu, X. Chen, Q. Wang, S. Guo, In situ Synthesis of Silver-nanoparticles/bacterial Cellulose Composites for Slow-released Antimicrobial Wound Dressing. Carbohydr. Polym. 102 (2014) 762-771. 9. P.A.Tran, D. M. Hocking, A.J. O’Connor, In situ Formation of Antimicrobial Silver Nanoparticles and the Impregnation of Hydrophobic Polycaprolactone Matrix for Antimicrobial Medical Device Applications. Matr. Sci Eng. C 47 (2015) 63-69. 10. S. Ferraris, S. Spriano, Antibacterial Titanium Surfaces for Medical Implants, Matr. Sci. Eng. C 61 (2016) 965-978. 11. Y.N. Slavin, J. Asnis, U.O. Hafeli, H. Bach, Metal Nanoparticles: Understanding the Mechanisms behind Antibacterial Activity, J. Nanobiotechnol. 15 (2017) 65. 12. K. Zodrow, L. Brunet, S. Mahendra, D. Li, A. Zhang, Q.L.Li, P.J.J. Alvarez, Polysulfone Ultrafiltration Membranes Impregnated with Silver Nanoparticles Show Improved Biofouling Resistance and Virus Removal, Water Res. 43 (2009) 715-723. 13. X.Y. Zhu, R.B. Bai, K.H. Wee, C.K. Liu, S.L. Tang, Membrane Surfaces Immobilized with Ionic or Reduced Silver and their Anti-biofouling Performances, J. Membr. Sci. 363 (2010) 278-286. 14. J.B.D. Green, T. Fulghum, M.A. Nordhaus, A Review of Immobilized Antimicrobial Agents and Methods for Testing, Biointerphases, 6 (2011) MR13-MR28. 15. X-F. Sun, J Qin, P-F. Xia, B.-B. Guo, C.-M. Yang, C. Song, S.-G. Wang, Graphene oxide-Silver Nanoparticle Membrane for Biofouling Control and Water Purification. Chem. Eng. J. 281 (2015) 53-59.



16. D. Rana, T. Matsuura, Surface Modifications for Antifouling Membranes, Chem. Rev., 110 (2010) 2448-2471. 17. P. Gunawan, C. Guan, X. Song, Q. Zhang, S.S.J. Leong, C. Tang, Y. Chen, M.B. ChanPark, M.W. Chang, K. Wang, R. Xu, Hollow Fiber Membrane Decorated with Ag/MWNTs: Toward Effective Water Disinfection and Biofouling Control, ACS Nano, 5 (2011) 10033-10040. 18. E.S. Kim, G. Hwang, M.G. El-Din, Y. Liu, Developmemnt of Nanosilver and Multiwalled-carbon Nanotubes Thin-film Nanocomposite Membrane for Enhanced Water Treatment. J. Membr. Sci. 394-395 (2012) 37-48. 19. S.Y. Lee, H.J. Kim, R. Patel, S.J. Im, J.H. Kim, B.R. Min, Silver Nanoparticles Immobilized on Thin Film Composite Polyamide Membrane: Characterization, Nanofiltration, Antifouling Properties, Polym. Adv. Technol. 18 (2007) 562-568. 20. X. Cao, M. Tang, F. Liu, Y. Nie, C. Zhao, Immobilization of Silver Nanoparticles onto Sulfonated Polyethersulfone Membranes as Antibacterial Materials, Colloids Surf. B: Biointerfaces 81 (2010) 555-562. 21. M.S. Mauter, Y.Wang, K.C. Okemgbo, C.O. Giannelis, M. Elimelech, Antifouling Ultrafiltration Membranes via Post-fabrication Grafting of Biocidal Nanomaterials, ACS Appl. Mater. Interfaces 3 (2011) 2861-2868. 22. J. Yin, Y. Yang, Z. Hu, B. Deng, Attachment of Silver Nanoparticles (AgNPs) onto ThinFilm Composite (TFC) Membranes through Covalent Bonding to Reduce Membrane Biofouling, J. Membr. Sci. 441 (2013) 73-82.


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23. J. Li, X. Liu, J. Lu, Y. Wang, G. Li, F. Zhao, Anti-bacterial Properties of Ultrafiltration Membrane Modified by Graphene Oxide with Nano-silver Particles, J. Colloid Interface Sci. 484 (2016) 107-115. 24. I. Sadeghi, H. Yi, A. Asatekin, A Method for Manufacturing Membranes with Ultrathin Hydrogel Selective Layers for Protein Purification: Interfacially Initiated Free Radical Polymerization (IIFRP), Chem. Mater., 30 (2018) 1265-1276. 25. P. Bahmani, A. Maleki, H. Daraei, M, Khamforoush, R. Rezaee, F. Gharibi, A. G. Tkachev, A.E. Burakov, S. Agarwal, V.K. Gupta, High-flux Ultrafiltration Membrane based on Electrospun Polyacrylonitrile Nanofibrous Scaffolds for Arsenate Removal from Aqueous Solutions, J. Colloid Interface Sci. 506 (2017) 564-571. 26. H. Ju, B.D. McCloskey, A.C. Sagle, Y.-H. Wu, V.A. Kusuma, B.D. Freeman, Crosslinked Poly(ethylene oxide) Fouling Resistant Coating Materials for Oil/water Separation, J. Membr. Sci. 307 (2008) 260-267. 27. G. Kang, Y. Cao, H. Zhao, Q. Yuan, Preparation and Characterization of Crosslinked Poly(ethylene glycol) Diacrylate Membranes with Excellent Antifouling and SolventResistant Properties, J. Membr. Sci. 318 (2008) 227-232. 28. X. Xhang, B. Lin, K. Zhao, J.Wei, J. Guo, W. Cui, S. Jiang, D. Liu, J. Li, A Freestanding Calcium Alginate/polyacrylamide Hydrogel Nanofiltration Membrane with High Anti-fouling Performance: Preparation and Characterization, Desalination 365 (2015) 234-241. 29. K.A. Rieger, M. Porter, J.D. Schiffman, Polyelectrolyte-functionalized Nanofiber Mats Control the Collection and Inactivation of Escherichia coli, Materials 9 (2016) E297. 28 ACS Paragon Plus Environment


30. K.M. Dobosz, C.A. Kuo-Leblanc, T.J. Martin, J.D. Schiffman, Ultrafiltration Membranes Enhanced with Electrospun Nanofibers Exhibit Improved Flux and Fouling Resistance, Ind. Eng. Chem. Res. 56 (2017) 5724-5733. 31. Y.-F. Zhao, L.-P. Zhu, Z. Yi, B.-K. Zhu, Y.-Y. Xu, Zwitterionic Hydrogel Thin Films as Antifouling Surface Layers of Polyethersulfone Ultrafiltration Membranes Anchored via Reactive Copolymer Additive, J. Membr. Sci., 470 (2014) 148-158. 32. S. M. Salehi, G. Di Profio, E. Fontananova, F. P. Nicoletta, E. Curcio, G. De Filpo, Membrane Distillation by Novel Hydrogel Composite Membranes, J. Membr. Sci., 504 (2016) 220-229. 33. M. He, Q. Wang, R. Wang, Y. Xie, W. Zhao, C. Zhao, Design of Antibacterial Poly(ether sulfone) Membranes via Covalently Attaching Hydrogel Thin Layers Loaded with Ag Nanoparticles, ACS Appl. Mater. Interfaces 9 (2017) 15962-15974. 34. H. Wang, G. Y. Zha, H. Du, L. L. Gao, X. D. Li, Z. Q. Shen, W. P. Zhu, Facile Fabrication of Ultrathin Antibacterial Hydrogel Films via layer-by-layer “Click” Chemistry, Polym. Chem., 5 (2014) 6489-6494. 35. M. He, Q. Wang, W. Zhao, C. Zhao, A Substrate-independent Ultrathin Hydrogel Film as an Antifouling and Antibacterial Layer for a Microfiltration Membrane Anchored via a layer-by-layer thiol-ene Click Reaction, J. Mater. Chem. B, 6 (2018) 3904-3913. 36. H. Zou, Q. Luo, D. Zhou, Affinity Membrane Chromatography for the Analysis and Purification of Proteins. J. Biochem. Biophys. Methods. 49 (2001) 199–240 37. K. G. Briefs, M.R. Kula, Fast Protein Chromatography on Analytical and Preparative Scale using Modified Microporous Membrane. Chem. Eng. Sci. 47 (1992) 141-149


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38. A. Kumari, L. Rekhi, S. Datta, Reversibly Attached Phospholipid Bilayer Functionalized Membrane Pores. Langmuir. 2018, DOI 10.1021/acs.langmuir.8b03404 39. C.M. Gonzalez-Henriquez, G. del C. Pizarro, M.A. Sarabia-Vallejos, C.A. Terraza, Z. E. Lopez-Cabana, In situ-preparation and Characterization of Silver-HEMA/PEGDA Hydrogel Matrix Nanocomposites: Silver Inclusion Studies into Hydrogel Matrix, Arab. J. Chem. (2014) 10.1016/j.arabjc.2014.11.012 40. N. Ahmad, S. Sharma, M.K. Alam, V.N. Singh, S.F. Shamsi, B.R. Mehta, A. Fatma, Rapid Synthesis of Silver Nanoparticles using Dried Medicinal Plant of Basil, Colloids Surf. B Biointerfaces 81 (2010) 81-86. 41. M. Sakamoto, M. Fujistuka, T. Majima, Light as a Construction Tool of Metal Synthesis: Synthesis and Mechanism, J. Photochem. Photobiol. C: Photochem. Rev. 10 (2009) 3356. 42. B. T. McVerry, T J. A. Temple, X. Huang, K. L. Marsh, E. M. V. Hoek, R. B. Kaner, Fabrication of Low-Fouling Ultrafiltration Membranes Using a Hydrophilic, Self-Doping Polyaniline Additive. Chem. Mater. 25 (2013) 3597−3602. 43. D. Y. Zhang, Q. Hao, J. Liu, Y. S. Shia, J. Zhu, L. Su, Wang, Y. Antifouling Polyimide Membrane with Grafted Silver Nanoparticles and Zwitterion. Sep. Purif. Technol., 192 (2018) 230–239 44. Y-H. La, B. D. McCloskey, R. Sooriyakumaran, A. Vora, B. Freeman, M. Nassar, J. Hedricka, A. Nelsona, Allen, R. Bifunctional Hydrogel Coatings for Water Purification Membranes: Improved Fouling Resistance and Antimicrobial Activity. J. Membr Sc. 372 (2011) 285–291 30 ACS Paragon Plus Environment



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In Situ Synthesis of Silver Nanoparticles within Hydrogel-conjugated

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