Novel Active Surface Prepared by Embedded Functionalized Clays in

Subscriber access provided by NEW YORK MED COLL

Article

Novel Active Surface Prepared by Embedded Functionalized Clays in an Acrylate Coating Yining Xia, Mehran Ghasemlou, Maria Rubino, Rafael Auras, and Jamil Baghdachi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08579 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

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

ACS Applied Materials & Interfaces

Novel Active Surface Prepared by Embedded Functionalized Clays in an Acrylate Coating Yining Xia1, Mehran Ghasemlou1, Maria Rubino1,*, Rafael Auras1and Jamil Baghdachi2 1

2

School of Packaging, Michigan State University, East Lansing, MI 48824, USA Coatings Research Institute, Eastern Michigan University, Ypsilanti, MI 48197, USA

ABSTRACT The research on self-decontaminating surface has received significant attention due to the growth of pathogenic microorganisms on surfaces. In this study, a novel and simple technique for producing an active surface with antimicrobial functionality is demonstrated. A tethering platform was developed by grafting the biocide ampicillin (Amp) to a nanoclay and dispersing the nanoclay in a UV-curable acrylate coating applied on polypropylene films as the substrate. A coupling agent, 3-glycidyloxypropyl trimethoxysilane, was used as a linker between the nanoclay and Amp. The Amp-functionalized clay was further modified with an organic surfactant to improve the compatibility with the coating. Several characterization assays, such as Fourier infrared transform analysis, thermogravimetric analysis and X-ray diffraction, were conducted to confirm the presence of Amp in the nanoclay. Transmission electron microscopy images revealed that the clay particles were well dispersed in the coating and had a partial exfoliated morphology. The active coating surface was effective in inhibiting the growth of gram-positive Listeria monocytogenes and gram-negative Salmonella Typhimurium via contact. These findings suggest the potential for the development of active surfaces with implementation of nanotechnology to achieve diverse functionalities.

KEYWORDS: active surface, nanoclay, ampicillin, covalent grafting, acrylate coating

1 ACS Paragon Plus Environment


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

INTRODUCTION Active ingredients, such as radical scavengers, UV-absorbing compounds, antioxidants and hygiene agents, find uses in a wide variety of consumer goods.1−3 A product surface incorporating one or more of these ingredients is considered an active surface, which acts as a potential barrier to undesirable conditions that compromise the product quality and safety. The active surface has become a subject of intensive research, with focus on surface functionalization to provide sufficient protection to the product.4−6 For example, antimicrobial functionality can be achieved by incorporating antimicrobial agent(s) as the active ingredient(s). Microbial attachment and subsequent growth of pathogenic microorganisms on surfaces is a serious concern to public health. To address this issue, considerable research efforts have aimed to create surfaces with microbial inhibition capability that can be applied to various commercial products such as packaging, children’s toys, hospital implants and surgical equipment. A number of different techniques have been explored to create active surfaces with antimicrobial properties. An active surface can be generated via the adsorption of active ingredients on the material surface, with a slow release of ingredients into the surrounding environment.7 Immobilization of active ingredients to the material surface through covalent grafting or ionic bonding is another approach to achieve antimicrobial functionality.8,9 This latter approach has long-term antimicrobial efficiency due to the lack of release of biocidal moieties into the environment; it also reduces human exposure to biocides that may cause adverse health effects or provide conditions for the development of bacterial resistance.10 Nanotechnology has received significant attention in the development of active surface by adding engineered nanoparticles (ENPs) to the surface matrix.11,12 Among the ENPs, nanoclays, such as organo-modified montmorillonite, are promising nanoparticles due to their


Page 2 of 23

Page 3 of 23

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


low cost, commercial availability and relatively simple processability. The addition of nanoclay to polymers results in materials with enhanced mechanical, thermal and barrier properties.13 In addition, the large surface area of a nanoclay makes it an ideal carrier for active ingredients by adsorbing or immobilizing the chemical compounds to the particle surface, which imparts novel functionality to the nanoparticle.14−16 Therefore, by utilizing nanotechnology, an active surface can be developed with both improved material performance and antimicrobial efficacy. Several processing techniques have been developed to confer substrates with nanoparticle-enabled active surfaces. Casting is a common technique to cover a thin layer of polymer film on a substrate. However, considering the need for high levels of film production and the environmental issues caused by the use of organic solvents, other technologies must be developed to fulfill these demands. Extrusion-based processing is one of the most successful technologies to manufacture a polymer substrate with an active surface because of its versatility, high productivity and low costs. Nevertheless, the high temperature and pressure involved in the processing procedures may result in considerable loss of the active ingredient, and limit the number of active ingredients that can be used. An alternative approach to overcome these challenges is to use a UV-curing technology to apply a coating or surface on a substrate. UVcurable coatings are well accepted in many industrial applications and are associated with several advantageous features such as high-speed processing, low energy consumption, minimum heat generated, environmental friendliness by avoiding solvent usage, and adaptability to different substrates with superior adhesion.17−20 The additional benefits of this technique are that the amount of active ingredient(s) loaded on a surface is considerably less than the amount normally added in the bulk material, and has no impact on the characteristics of the bulk material.



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

This paper presents a novel approach for the development of an active surface by homogenously dispersing biocide-functionalized clay in a UV-curable coating. Ampicillin (Amp), a biocide with broad-spectrum antimicrobial activity, was served as a model molecule to functionalize the nanoclay via covalent grafting, and was dispersed in the coating along with the nanoclay. The coating was applied on a substrate (polypropylene film), and its antimicrobial activity was characterized and evaluated. The outcome of this study indicates the feasibility of developing active surfaces by implementing nanotechnology, especially the use of functionalized nanoparticles.

MATERIALS AND METHODS Materials. Polymer grade montmorillonite (MMT) was obtained from Nanocor (Aberdeen, MS, U.S.A.). The coupling agent 3-glycidyloxypropyl trimethoxysilane (3GTS) was purchased from Sigma-Aldrich (boiling point =120°C, MW = 236 Da). N,N-bis(2hydroxyethyl)-N-methyl-N-tallow ammonium chloride (or TomamineTM Q-T-2 70% PG) at 60– 70% purity with 30–40% propylene glycol was used as an organo-modifier (surfactant) for the nanoclay and was obtained from Air Products and Chemicals Inc. (Butler, IN, U.S.A.). Ampicillin (Amp) (>96% purity, melt point 208°C, MW = 349 Da) was purchased from SigmaAldrich; Amp is insoluble in alcohol, slightly soluble in water, and more soluble in dilute acids or bases. The acrylate coating formula was provided by Sartomer (Exton, PA, U.S.A.) and consists of acrylate monomers, acrylate oligomers, a photoinitiator and a wetting agent (detailed information is provided in the Supporting Information, Table S1). PP resin (Profax 6523) was supplied by LyondellBasell Industries (Houston, TX, U.S.A.) and was converted to films via blow film extrusion as described in a previous study.21


Page 4 of 23

Page 5 of 23

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


Preparation of Functionalized Clay. Figure 1 shows the method used to covalently graft Amp to the nanoclay in a two-reaction process. 3GTS was used as a bridge in the first step to link Amp (amine group) with the epoxy-terminated side chain, and then in the second step to link the clay surface (silanol group) with the methoxy group. Briefly, 1 g of Amp and 0.4 g of 3GTS were mixed together in a beaker filled with 100 mL of water and 0.001 M acetic acid (pH ≈ 4), and the mixture was stirred at 40°C for 2 h. In a separate beaker, a nanoclay suspension was prepared by adding 1.5 g of MMT clay into 50 mL of methanol and stirring the mixture at room temperature for 15 min. The nanoclay suspension was then gradually added into the pretreated Amp-3GTS mixture, and the mixture was stirred for another 4 h at 40°C. After the reaction, the functionalized clay was washed with hot water and centrifuged (5000 rpm, 10 min) at least six times, and then vacuum dried at room temperature. To ensure that the free Amp was removed from the nanoclay, the absorbance at 225 nm of the washing solution at each washing cycle was checked with a UV-Vis spectrometer until no detectable amount of Amp was presented in the solution (detailed method is described in the Supporting Information). To improve the compatibility of the nanoclay with the acrylate coating, the Amp-grafted clay (designated as MMT-g-Amp) was organo-modified with the Tomamine surfactant (clay: surfactant =1: 1, w/w) through ion exchange in water stirred for 8 hat room temperature. The functionalized clay after organo-modification (designated as OMMT-g-Amp) was washed with water at least three times, centrifuged (5000 rpm, 5 min) and then vacuum dried at room temperature. MMT clay modified with the Tomamine surfactant (designated as OMMT) was also prepared in the same manner.



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

Figure 1. Method for grafting ampicillin to nanoclay.

Preparation of Active Coating. Acrylate coatings with functionalized clay (designated as COA-OMMT-g-Amp) at three different concentration levels (1, 3 and 5 wt%) were prepared. The functionalized clay at each concentration level was first mixed with acrylate monomers in a ceramic mortar. The clay-monomer mixture was transferred to a clear glass vial and sonicated in a Model FS30 ultrasonic cleaner (35 kHz, Fisher Scientific Co., Pittsburg, PA, U.S.A.) for 15 min to allow swelling of the functionalized clay. The other coating components, consisting of acrylate oligomers, photoinitiator and wetting agent, were then added and mixed with a spatula. A control coating containing 5 wt% OMMT was also prepared in the same manner. The acrylate coating was applied on the PP film substrate. The PP film surface was pre-treated with oxygen plasma (Plasma Science PS500 RF, AST Products, Inc., Billerica, MA, U.S.A.) for 30 s to achieve a surface energy of 45 mN m−1, in order to improve the adhesion between the acrylate coating and the PP film. The coating formula (0.5 mil or 13 µm in thickness) was then spread on 6 ACS Paragon Plus Environment

Page 6 of 23

Page 7 of 23

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


the treated film with a spiral bar type automatic film applicator, and cured at room temperature for about 4 s by using a Fusion 300S UV curing conveyor system (Fusion UV Systems Inc., Gaithersburg, MD, U.S.A.) equipped with a 1.8 kW, 6-inch UV lamp (main wavelength: 340 nm). Fourier Transform Infrared (FTIR) Spectroscopy. The infrared spectrum of the pristine clay (MMT) as well as the Amp-grafted clay (MMT-g-Amp) was recorded by a Shimadzu IR-Prestige 21 spectrometer (Shimadzu Co., Columbia, MD, U.S.A.) with an attenuated total reflection (ATR) accessory (PIKE Technologies, Madison, WI, U.S.A.). Sixtyfour (64) scans were done for each sample, with a spectral range of 400 to 4000 cm−1 and a resolution of 4 cm−1. Thermogravimetric Analysis. The weight loss of the pristine clay (MMT) as well as the functionalized clay (MMT-g-Amp and OMMT-g-Amp) as a function of temperature was determined by thermogravimetric analysis (TGA) with a Q-50 thermogravimetric analyzer (TA Instruments, New Castle, DE, U.S.A.). The sample was heated from 23 to 700 °C at a constant ramp rate of 10 °C min−1 under nitrogen flow (flow rate 70 mL min−1). X-ray Diffraction. X-ray diffraction (XRD) analysis was performed with a Bruker AXS D8 Advance X-ray diffractometer (Bruker Co., Billerica, MA, U.S.A.) equipped with a Global Mirror filtered Cu Kα radiation source (wavelength = 0.1542 nm) setting at 40 kV and 40 mA. The active coating sample as well as the clay powders (pristine and functionalized were scanned at a rate of 0.5 ° min−1 with a diffraction angle (2θ) range of 1 to 10 ° and an increment of 0.02 °. Transmission Electron Microscopy. Transmission electron microscopy (TEM) images were obtained with a JEOL 100CX II TEM instrument (JEOL USA Inc., MA, USA) at an accelerating voltage of 120 kV. The active coating samples were embedded in a paraffin block



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

and cut with a diamond knife on a RMC ultra-microtome (RMC, MT-7000, Tucson, AZ, U.S.A.). Microtomed coating specimens with thickness of less than 100 nm were placed on a copper grid and observed in bright field imaging mode of the instrument. Bacterial Adhesion Test. To assess the antimicrobial activity of the coating in both a qualitative and quantitative manner, an in vitro antibacterial test was performed in accordance with ASTM methods E2180 and JIS Z 2801. Two different bacteria, gram-positive Listeria monocytogenes and gram-negative Salmonella Typhimurium, were used (information regarding the preparation of the bacterial strains is provided in the Supporting Information). Samples of acrylate coating with functionalized clay (1, 3 and 5 wt%) and the control coating (with 5 wt% OMMT) were cut and placed individually in a separate Petri dish. Bacterial suspension of 10 µL (approximately 106 CFU/mL, CFU=colony forming units) was spread over each coating sample, the samples were then covered with a piece of sterile PP film, and the plates were incubated at 37 °C for 24 h. After incubation, each sample was removed, transferred into a tube containing phosphate buffer solution (PBS) and vortexed to detach the bacteria from the coating surface. After serial dilutions in PBS, the bacterial suspension at each dilution was plated (in triplicate) on trypticase soy agar (TSA) for S. Typhimurium and on modified Oxford agar (MOX) for L. monocytogenes, and incubated at 37 °C for 24 h to allow the growth of the bacteria. The number of bacterial colonies was then counted and compared. All the coating samples were tested in triplicate.

Agar Diffusion Test. An agar diffusion test was carried out to evaluate the inhibitory zone, if any, caused by the release of Amp into the agar. The active coating samples containing 5 wt% functionalized clay (in triplicate) were aseptically cut into discs (6-mm diameter) with a


Page 8 of 23

Page 9 of 23

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


sterile punch. The discs were placed on plates containing TSA that were previously seeded with 100 µL of overnight broth culture with a bacteria population of approximately 106 CFU mL−1. The plates were incubated at 37 °C for 24 h. Statistical Analysis. The antimicrobial results were analyzed using one-way analysis of variance (ANOVA) in the SAS program (Version 9.4; Statistical Analysis System Institute Inc., Cary, NC, U.S.A.). Differences between the mean values of bacterial counts were compared using Tukey's multiple range tests with a significance level of α = 0.05.

RESULTS AND DISCUSSION Characteristics of Functionalized Clay. Fourier infrared transform (FTIR) analysis was employed to confirm the functionalization of nanoclay with Amp. Figure 2 shows the FTIR spectrum of Amp-functionalized clay in the range of 1000 to 4000 cm−1, which exhibits several characteristic bands originated from the Amp molecule. A peak at 1764 cm−1 is associated with the stretching vibration of C=O in the β-lactam ring of Amp, and a peak at 1680 cm−1 represents the amide carbonyl group.22 The two peaks at 1505 cm−1 and 1460 cm−1are associated with the C=C stretching vibration of the aromatic ring. The vibrational band observed at1300 cm−1 is assigned to the C-N stretching mode of the amine group in the Amp molecule. Considering that the free Amp was washed out during the preparation procedures (see Supporting Information, Figure S1, for the decay of signal with washing cycle), the Amp detected by FTIR analysis was mostly grafted to the nanoclay.



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

Figure 2. FTIR spectra of pristine clay (MMT) and Amp-functionalized clay (MMT-gAmp).The inset has the same axis units as the larger graph.

Thermogravimetric analysis (TGA) was used to characterize the thermal stability of functionalized clay and measure the amount of Amp in the nanoclay. Figure 3 shows the TGA thermograms for the pristine clay as well as the functionalized clay without and with surfactant. An initial weight loss was observed before 100 °C due to the evaporation of water. The weight loss in the temperature range of 100 to 550 °C was mainly attributed to the decomposition of organic compounds (3GTS, Amp and surfactant) whereas decomposition of surfactant only occurred after 200 °C.23 The functionalized clay with surfactant showed lower weight loss (2%). The presence of surfactant appeared to improve the thermal stability of the grafting compound (3GTS-Amp) and thereby deferred its decomposition. The weight remaining at 550 °C was assumed to be the amount of MMT, and a small portion of weight loss after this temperature was caused by the dehydroxylation of MMT.23 Based on the TGA thermograms, the


Page 10 of 23

Page 11 of 23

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


composition of each type of clay was estimated (Table 1). The Amp content was 4.9 wt% in MMT-g-Amp and 3.6 wt% in OMMT-g-Amp. These values were high enough to expect antimicrobial activity of the functionalized clay when applied on a surface in contact with bacteria, as Amp is a powerful antimicrobial agent.24

Figure 3. Thermogravimetric profiles of the pristine clay (MMT) and the functionalized clay without surfactant (MMT-g-AMP) and with surfactant (OMMT-g-Amp).

Table 1. Composition in wt% of the pristine and functionalized clays. Clay components, wt% Sample Water

3GTS-Amp

3GTS

Amp

Surfactant

MMT

MMT

8.3

0

0

0

0

91.7

MMT-g-Amp

9.4

8.2

3.3

4.9

0

82.4

OMMT-g-Amp

1.9

6.1

2.5

3.6

31.4

60.6

Note: The 3GTS-Amp content in MMT-g-Amp was estimated based on the weight loss at 100 to



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

550 °C, then 3GTS and Amp contents were calculated based on their molecular weight ratio (3GTS:Amp = 236:349). The 3GTS-Amp content in OMMT-g-Amp was calculated based on the mass ratio of 3GTS-Amp to MMT for MMT-g-Amp (8.2:82.4).

Structure and Morphology. Good compatibility between the nanoclay and the acrylate coating can be achieved by selecting the appropriate organic surfactant for the nanoclay. In this study, the clay layers were easily intercalated by the coating formula and even exfoliated into the coating matrix. The structure and morphology of functionalized clay in the coating were evaluated by the combination of XRD and TEM. As shown by the XRD patterns (Figure 4a), the pristine clay (MMT) exhibited a diffraction peak at approximately 2θ = 7.31 ° (d= 1.21 nm), corresponding to the basal interlayer spacing.25 Upon functionalization with Amp, a shift in XRD pattern was observed from 2θ = 7.31 ° to 2θ = 5.70 ° (d = 1.55 nm). These values suggest that 3GTS-Amp intercalated into the clay gallery and reacted with the silanol groups on the clay surface. A significant increase in d-spacing from 1.55 nm to 3.78 nm appeared after the intercalation of surfactant into the clay gallery. A further increase of d-spacing to 4.21 nm was observed when the nanoclay was embedded in the coating (COA-OMMT-g-Amp), suggesting a slight swelling of nanoclay by the coating formula. TEM in conjunction with XRD analysis showed that the nanoclay was homogeneously dispersed in the coating matrix with an intermediate morphology between intercalation and exfoliation (Figure 4b). A broad distribution of particle sizes, from tens to hundreds of nm, was observed. A good dispersion of nanoclay ensured a homogeneous dispersion of Amp (as grafted to the nanoclay) throughout the coating, which could improve the antimicrobial efficacy of the coating.


Page 12 of 23

Page 13 of 23

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


Figure 4. (a) XRD patterns of nanoclay before and after functionalization, and (b) TEM image of functionalized clay in the coating.

Antimicrobial activity. A bacterial adhesion test was conducted to provide qualitative and quantitative information on the antimicrobial activity of the coating. Figure 5 shows the images for the antimicrobial activity of the coating against L. monocytogenes and S. Typhimurium. All the coating samples containing functionalized clay (COA-OMMT-g-Amp) showed antimicrobial activity against both bacteria as compared with the control coating: fewer colonies survived on the agar plates with functionalized clay coating samples, and the number of colonies decreased as the amount of functionalized clay increased in the coating.



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

Figure 5. Colonies of L. monocytogenes on MOX agar and S. Typhimurium on TSA after 24-h contact with the coating surface containing a different amount (wt%) of functionalized clay.

The antimicrobial activity was also quantitatively investigated by comparing the reduction of viable bacteria after contact with the different coating samples (Table 2). The reduction was strongly dependent on the loading of functionalized clay and the type of bacteria. A relatively low antimicrobial activity (0.11 log CFU mL−1 reduction) compared with the control was observed for L. monocytogenes on the coating containing 1 wt% functionalized clay, while a maximum reduction of 1.75 log CFU mL−1 was found at a clay loading of 5 wt%. At present, to the authors’ knowledge, no existing active surface can reduce bacterial attachment to an acceptable level or completely eliminate bacteria, and this is a major challenge in biological science.26 Several strategies have been proposed to reduce the extent of contamination on active surfaces and most were partially successful, reporting reductions up to 1 to 2 orders of magnitude.27 Since high levels of contamination are unlikely to happen, this range of reduction could still be considered effective from a public health viewpoint.28 The antimicrobial activity of the coating was lower for S. Typhimurium than for L. monocytogenes as indicated by the higher


Page 14 of 23

Page 15 of 23

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


population of S. Typhimurium at each clay loading; a maximum reduction of 0.60 log CFU mL−1 was found at the highest clay loading (5 wt%). This initial results revealed lower sensitivity of gram-negative bacteria than gram-positive bacteria because of an additional external membrane surrounding the cell wall in gram-negative bacteria, which promotes their resistance to the antimicrobial agents.29 It should be noticed that the grafting procedures caused a structural change on the Amp molecule by converting the primary amine (amino group) to secondary amine. Such change may affect the antimicrobial activity of grafted Amp in comparison to its isolated state since the amino group helps Amp to penetrate the outer membrane of bacteria and kill the bacteria.30 However, the remaining part of the Amp molecule (except the amino group) is comparable to many other members in the penicillin or aminopenicillin family, and it is still effective against bacteria.

Table 2. Populations of L. monocytogenes and S. Typhimurium after 24-h contact with active acrylate coating containing various levels (wt%) of functionalized clay. Listeria

Salmonella

Clay content CFU mL−1

Log CFU mL−1

CFU mL−1

Log CFU mL−1

Controla

(3.07 ± 0.10)×108

8.49 ± 0.01bA

(2.61 ± 0.24)×109

9.42 ± 0.04A

1 wt%

(2.40 ± 0.11)×108

8.38 ± 0.02A

(1.36 ± 0.09)×109

9.13 ± 0.03B

3 wt%

(2.06 ± 0.17)×107

7.31 ± 0.04B

(1.12 ± 0.08)×109

9.05 ± 0.03B

5 wt%

(5.57 ± 1.12)×106

6.74 ± 0.09C

(6.73 ± 1.37)×108

8.82 ± 0.09C

Note: aControl refers to the coating containing 5 wt% OMMT.bValues are expressed as mean ± standard deviation, and means within each column with different uppercase letters are significantly different (p< 0.05, n = 3).



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 addition to the bacterial adhesion test, an agar diffusion test was conducted on the coating samples against L. monocytogenes and S. Typhimurium. No zone of inhibition (Figure 6) was observed for either bacterial strain tested, indicating no release of Amp from the coating into the surrounding agar. The results of the agar diffusion test suggest that Amp was immobilized to the nanoclay dispersed in the coating, forming a self-decontaminating surface. Such a surface was effective in inhibiting the growth of bacteria through contact killing, which was demonstrated by the bacterial adhesion test.

Figure 6. Agar diffusion test of the coating (as indicated by the arrows) containing 5 wt% functionalized clay against (a) L. monocytogenes and (b) S. Typhimurium.

CONCLUSIONS We reported a novel approach to create an active surface with implementation of nanotechnology to achieve antimicrobial activity. Two essential features in the design of the active surface were the use of nanoclay and a UV-curable coating. The nanoclay was functionalized by grafting the biocide ampicillin to the clay surface with a silane compound as the linker. The functionalized clay was modified by an organic surfactant and then homogenously dispersed in an UV-curable


Page 16 of 23

Page 17 of 23

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


acrylate coating. The immobilization of Amp to the nanoclay was verified by instrumental analysis and microbiological assessment (agar diffusion test), which indicated the absence of free Amp in the coating. The coating was easily applied to a substrate through current industrial processing techniques to form an active surface. The antimicrobial activity of Amp was not adversely affected by its grafting to the nanoclay, and the fabricated surface containing Ampfunctionalized clay was effective in inhibiting the growth of L. monocytogenes and S. Typhimurium via contact. As a template, the nanoclay can be functionalized not only with ampicillin but also with many other active ingredients by using the same procedures in this study, so diverse functionalities can be brought to the active surface. Therefore, the proposed approach is not limited to creating an active surface for antimicrobial application, but can be expanded to other applications with high efficiency and flexibility.

ASSOCIATED CONTENT Supporting Information Composition of the acrylate coating, preparation of bacterial strains, residual check of ampicillin in the washing solution. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; Tel: +1(517)355-0172.

ACKNOWLEDGEMENTS



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

This work was financially supported by the Center for Packaging Innovation and Sustainability at Michigan State University and by the USDA National Institute of Food and Agriculture and Michigan AgBioResearch, Hatch projects M. Rubino and R. Auras. The authors are grateful to Prof. Elloit Ryser from the Department of Food Science and Human Nutrition for assistance with microbial analysis and access to the microbiology lab.

REFERENCES (1) Kumar, A.; Vemula, P. K.; Ajayan, P. M.; John, G. Silver-nanoparticle-embedded antimicrobial paints based on vegetable oil. Nat. Mater. 2008, 7, 236−241. (2) Lacatusu, I.; Badea, N.; Murariu, A.; Meghea, A. The encapsulation effect of UV molecular absorbers into biocompatible lipid nanoparticles. Nanoscale Res. Lett. 2011, 6, 73−81. (3) Kanatt, S. R.; Rao, M. S.; Chawla, S. P.; Sharma, A. Active chitosan–polyvinyl alcohol films with natural extracts. Food Hydrocolloids 2012, 29, 290−297. (4) Tian, F.; Decker, E. A.; Goddard, J. M. Control of lipid oxidation by nonmigratory active packaging films prepared by photoinitiated graft polymerization. J. Agric. Food Chem. 2012, 60, 7710–7718. (5) Xiao, G.; Zhang, X.; Zhao, Y.; Su, H.; Tan, T. The behavior of active bacterial and antifungal coating under visible light irradiation. Appl. Surf. Sci. 2014, 292, 756–763. (6) Rels R.; Dumee, L. F.; He, L.; She, F.; Orbell, J. D.; Winther-Jensen, B.; Duke, M. C. Amine enrichment of thin-film composite membranes via low pressure plasma polymerization for antimicrobial adhesion. ACS Appl. Mater. Interfaces 2015, 7, 14644–14653. (7) Li, Z.; Lee, D.; Sheng, X.; Cohen, R. E.; Rubner, M. F. Two-level antibacterial coating with both release-killing and contact-killing capabilities. Langmuir 2006, 22, 9820−9823.


Page 18 of 23

Page 19 of 23

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) Green, J. B. D.; Fulghum, T.; Nordhaus, M. A. Immobilized antimicrobial agents: A critical perspective. Chem. Rev. 2009, 109, 5437-5527. (9) Liu, R.; Zheng, J.; Li, Z.; Liu, J.; Liu, X. Preparation of surface self-concentration and contact-killing antibacterial coating through UV curing. RSC Adv. 2015, 5, 34199−34205. (10) Agnihotri, S.; Mukherji, S.; Mukherji, S. Immobilized silver nanoparticles enhance contact killing and show highest efficacy: elucidation of the mechanism of bactericidal action of silver. Nanoscale 2013, 5, 7328−7340. (11) Neethirajan, S.; Jayas, D. S. Nanotechnology for the food and bioprocessing industries. Food Bioprocess Technol. 2011, 4, 39−47. (12) Llorens, A.; Lloret, E.; Picouet, P. A.; Trbojevich, R.; Fernandez, A. Metallic-based micro and nanocomposites in food contact materials and active food packaging. Trends Food Sci. Technol. 2012, 24, 19−29. (13) Sinha Ray, S.; Okamoto, M. Polymer/layered silicate nano-composites: a review from preparation to processing. Prog. Polym. Sci. 2003, 28, 1539−1641. (14) Haloi, D. J.; Ata, S.; Singha, N. K. Synthesis and characterization of all acrylic block copolymer/clay nanocomposites prepared via surface initiated atom transfer radical polymerization (SI-ATRP). Ind. Eng. Chem. Res. 2012, 51, 9760−9768. (15) Lvov, Y.; Abdullayev, E. Functional polymer–clay nanotube composites with sustained release of chemical agents. Prog. Polym. Sci. 2013, 38, 1690−1719. (16) Ibarguren, C.; Naranjo, P. M.; Stotzel, C.; Audisio, M. C.; Sham, E. L.; Torres, E. M. F.; Muller, F. A. Adsorption of nisin on raw montmorillonite. Appl. Clay Sci. 2014, 90, 88−95.



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

(17) Masson, F.; Decker, C.; Jaworek, T.; Schwalm, R. UV-Radiation curing of waterbased urethane–acrylate coatings. Prog. Org. Coat. 2000, 39, 115−126. (18) Bose, S.; Bogner, R. H. Solventless pharmaceutical coating processes: a review. Pharm. Dev. Technol. 2007, 12, 115−131. (19) Kim, D.; Jang, M.; Seo, J.; Nam, K. H.; Han, H.; Khan, S. B. UV-cured poly (urethane acrylate) composite films containing surface-modified tetrapod ZnO whiskers. Compos. Sci. Technol. 2013, 75, 84−92. (20) Maurin, V.; Croutxé-Barghorn, C.; Allonas, X.; Brendlé, J.; Bessières, J.; Merlin, A.; Masson, E. UV powder coatings containing synthetic Ag-beidellite for antibacterial properties. Appl. Clay Sci. 2014, 96, 73−80. (21) Xia, Y.; Rubino, M.; Auras, R. Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant. Environ. Sci. Technol. 2014, 48, 13617–13624. (22) Khan, E.; Shukla, A.; Srivastava, A.; Rao, S.; Tandon, P. Molecular structure, spectral analysis and hydrogen bonding analysis of ampicillin trihydrate: A combined DFT and AIM approach. New J. Chem. 2015, 10.1039/C5NJ01779C. (23) Cervantes-Uc, J. M.; Cauich-Rodriguez, J. V.; Vazquez-Torres, H.; Garfias-Mesias, L. F.; Paul, D. R. Thermal degradation of commercially available organoclays studied by TGAFTIR. Thermochim. Acta 2007, 457, 92−102. (24) Forestier, F.; Gerrier, P.; Chaumanrd, C.; Quero, A. M.; Couvreur, P.; Labarre, C. Effect of nanoparticle-bound ampicillin on the survival of Listeria monocytogenes in mouse peritoneal macrophages. J. Antimicrob. Chemother. 1992, 30, 173-179.


Page 20 of 23

Page 21 of 23

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


(25) Yei, D. R.; Kuo, S. W.; Su, Y. C.; Chang, F. C. Enhanced thermal properties of PS nanocomposites formed from inorganic POSS-treated montmorillonite. Polymer 2004, 45, 2633–2640. (26) Wimley, W. C.; Hristova, K. A look at arginine in membranes. J. Membr. Biol. 2011, 239, 49–56. (27) Privett, B. J.; Youn, J.; Hong, S.A.; Lee, J.; Han, J.; Shin, J. H.; Schoenfisch, M.H. Antibacterial fluorinated silica colloid superhydrophobic surfaces. Langmuir 2011, 27, 9597−9601. (28) Todd, E. C. Microbiological safety standards and public health goals to reduce foodborne disease. Meat Sci. 2004, 66, 33–43. (29) Kreutzwiesner, E.; Noormofidi, N.; Wiesbrock, F.; Kern, W.; Rametsteiner, K.; Stelzer, F.; Slugovc, C. Contact bactericides and fungicides on the basis of amino-functionalized poly(norbornene)s. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 4504−4514. (30) Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta, Proteins Proteomics 2009, 1794, 808−816.

TOC graphic



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


Page 22 of 23

Page 23 of 23

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


525x223mm (72 x 72 DPI)

ACS Paragon Plus Environment

Novel Active Surface Prepared by Embedded Functionalized Clays in

Recommend Documents