Polymeric Antimicrobial N-Halamine-Surface Modification of Stainless

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Polymeric Antimicrobial N-halamine-Surface Modification of Stainless Steel Buket Demir, R. M. Broughton, Tung-Shi Huang, Michael Bozack, and S. D. Worley Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b02412 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Industrial & Engineering Chemistry Research

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Polymeric Antimicrobial N-halamine-Surface Modification of Stainless

2

Steel

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Buket Demir,1 R. M. Broughton,2 T.S. Huang,3 M. J. Bozack,4 and S.D. Worley*,1

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1

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36849

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2

7

Engineering, Auburn University, Auburn, AL, 36849

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3

Department of Poultry Science, Auburn University, Auburn, Alabama 36849

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4

Department of Physics, Auburn University, Auburn, Alabama 36849

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*

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6980

Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama

Center for Polymers and Advanced Composites, Department of Mechanical

Author to whom correspondence should be addressed. [email protected] 334-844-

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ABSTRACT: The surfaces of materials fabricated from stainless steel are challenging

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to functionalize with antimicrobial moieties. This work demonstrates that stainless steel

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surfaces can be modified with an N-halamine-based copolymer in order to obtain

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antimicrobial activity. In this regard a copolymer (HACM) of 2-acrylamido-2-methyl-1-

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(5-methylhydantoinyl)propane and 3-cloro-2-hydroxypropyl methacrylate was

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synthesized and grafted onto a stainless steel surface via covalent attachment.

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Synthesized monomers and copolymers were characterized by NMR, FTIR, and XPS

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spectral analyses. Upon treatment with dilute bleach, the stainless steel surfaces were

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rendered antimicrobial, possessing a sufficient amount of chlorine content and excellent

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stability and durability. The modified stainless steel samples inactivated 6 logs of

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Staphylococcus aureus and Escherichia coli O157:H7 bacteria within 15 min of contact

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time. Stabilities of the coatings toward washing and UVA exposure were also studied.

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The stainless steel samples showed superior washing stabilities and re-generabilities.

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After 5 cycles of washing, there was a very minimal change in the initial chlorine

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contents, and the chlorine content could be recharged to its initial number of Cl+

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atoms/cm2. However, only a moderate stability of the coatings was observed after UVA

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irradiation. These results indicate that N-halamine precursor polymers can be facilely

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applied to stainless steel surfaces by covalent bonding, and that robust, re-generable

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antimicrobial stainless steel surfaces could be prepared via the N-halamine technology.

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This technology exhibits potential for use in food processing, prevention of biofilm

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formation, and in biomedical and health-care industries to support the prevention and

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reduction of cross-contamination and health-care related infections.

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KEYWORDS: N-halamines, antimicrobials, stainless steel, food processing, health

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care

37 38 39 40


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INTRODUCTION Prevention of cross contamination of microorganisms and microbial biofilm

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formation on surfaces employed in water purification and delivery systems, food

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processing equipment, hospital equipment, biomedical implants, and medical devices has

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been a major concern in the food and biomedical industries.1-3 Stainless steel has been

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widely used in these industries due to its mechanical properties and resistance to

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corrosion and chemicals.4,5 However, it is difficult to prevent adhesion and growth of

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bacteria, formation of stable biofilms, and the increased resistance to sanitization

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methods when stainless steel is used for surface applications in food processing plants

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and for biomedical applications. Microbial contamination is a source of chronic

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infections and the transmission of infectious diseases, and as such, it raises major public

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health risks in these industries.6 In addition, due to the negative impact on public health,

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it causes economic and product losses. Current methods for biofilm removal require

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usage of chemical disinfectants and heat. However, the methods do not prevent or control

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bacteria growth and biofilm formation, and they are ineffective, expensive, and raise

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environmental concerns.3 The chemical functionalization of stainless steel surfaces has

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proven to be a challenging endeavor. A review of this subject as relates to biofouling in

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the food industry has been presented.7 Therefore, there is a need for development of

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antimicrobial stainless steel surfaces to prevent cross contamination, to reduce

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microorganism adhesion and survival, and to inactivate microorganisms on stainless steel

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surfaces.

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Researchers have been exploring various surface modification strategies such as

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deposition of biocidal metals like silver, copper, and nickel,8-10 biopolymers,9,11-13 or

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inorganic oxides such as TiO2 anatase14 by electrolysis, plasma, chemical vapor

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deposition, layer-by-layer assembly by charged polyelectrolytes, electroless nickel, or

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sol-gel procedures on stainless steel surfaces.8-14 Also, encouraging studies have been

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reported concerning covalent attachment of biopolymers and synthetic polymers15 as well

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as antibacterial peptides bound to a chitosan layer16 for the prevention of bio-adhesion.

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However, further development of antimicrobial materials for surface modification that

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possesses long lasting, stable, non-toxic, and effective antimicrobial properties is 3 ACS Paragon Plus Environment


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necessary. In general the strategies mentioned above are expensive to implement, and

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contact times necessary to effect even modest log reductions of bacteria on stainless steel

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tend to be hours, which render practical application questionable.

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A class of biocidal materials known as the N-halamines has been employed in

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recent times for rendering a variety of surfaces antimicrobial.17-19 Among the many

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biocidal agents which have been employed for surfaces, N-halamines are generally the

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most effective due to superior antimicrobial efficacies against a broad spectrum of

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microorganisms in brief contact times (minutes), non-toxicities, stabilities, and re-

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generabilities.17-21 Although much of the work has been performed in the laboratories of

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the authors on a variety of novel antimicrobial N-halamine derivatives used in many

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different applications such as in water disinfection and in antimicrobial treatment of

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textiles,20,22-29 the field has been expanding into many other laboratories and

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applications.21,30-37

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To our knowledge the only work reported to date concerning N-halamine

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antimicrobial coatings on stainless steel has originated from the laboratory of Goddard

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and coworkers.37 In their work well characterized multilayer coatings of branched

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poly(ethylene imine) and poly(acrylic acid) were covalently attached to the surface of

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stainless steel by layer by layer deposition using a non-antimicrobial coupling layer to

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provide the grafting of the antimicrobial layer. Upon chlorination to produce an N-

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chloramine coating, the surfaces produced about a 1.5 log inactivation of Listeria

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monocytogenes at a contact time of 6 h.37

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In the current work a copolymer (HACM) of 2-acrylamido-2-methyl-1-(5-

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methylhydantoinyl) propane and 3-cloro-2-hydroxypropyl methacrylate was synthesized

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and covalently attached to stainless steel (see Scheme 1). Following chlorination with

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dilute bleach the surfaces demonstrated excellent antimicrobial efficacies (6 logs) against

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Staphylococcus aureus and Escherichia coli O157:H7 in contact times of 15 to 30 min. It

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has also been shown that upon washing, the surfaces maintain adequate stabilities of the

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oxidative chlorine and can be recharged following exhaustion of the chlorine. The work

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illustrates the potential for N-halamine modification of stainless steel to create an 4 ACS Paragon Plus Environment

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antimicrobial surface for a variety of possible applications in the food and biomedical

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industries.

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EXPERIMENTAL SECTION

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Materials and Instrumentation. 2-acrylamido-2-methyl-4-pentanone was

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purchased from Tokyo Chemical Industry, Co., LTD (Tokyo, JP). 3-cloro-2-

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hydroxypropyl methacrylate (CM), potassium cyanide, and ammonium carbonate were

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obtained from Acros Organics (NJ, USA). 2,2’-Azobis(2-methylpropionitrile) (AIBN)

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was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). All of the other chemicals

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were purchased from Aldrich Chemical Company (Milwaukee, WI), TCI America

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(Boston, MA), or Alfa Aesar (Heysham, UK), and used as received unless otherwise

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noted. Stainless steel 316L (ASTM A240/A240M standard) with 2B finish (Ra roughness

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of the surface typically being between 0.3 -0.4 µm) was purchased from McMaster-Carr

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(Chicago, IL). Clorox® brand (Clorox, Inc., Oakland, CA) household bleach (8.25%

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NaOCl) was used for the chlorination process. Bacterial cultures of Staphylococcus

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aureus ATCC 6538 and Escherichia coli O157:H7 ATCC 43895 were purchased from

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American Type Culture Collection (Rockville, MD), and Trypticase soy agar was

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obtained from Difco Laboratories (Detroit, MI).

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ATR-IR data were recorded with 32 scans at 4 cm-1 resolution with an ATR-FT-

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IR spectrometer (Model Spectrum 400, Perkin Elmer Co., Waltham, MA, USA). 1H

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NMR spectra were obtained with a Bruker 400 MHz spectrometer (16 scans).

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Photoemission measurements were performed with a load-locked Kratos XSAM 800

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surface analysis system equipped with a hemispherical energy analyzer. UVA stability

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testing was conducted using a Q-Panel accelerated weathering tester which provides

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UVA irradiation in the 315-400 nm regions.

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Synthesis of the HACM Copolymer for Stainless Steel Surface Modification.

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2-acrylamido-2-methyl-1-(5-methylhydantoinyl)propane (HA) was synthesized according

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to a protocol described previously (Supporting information, Figure S1).23 Briefly, 2-

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acrylamido-2-methyl-4-pentanone (0.1 mol), potassium cyanide (0.2 mol), and 5 ACS Paragon Plus Environment


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ammonium carbonate (0.6 mol) were reacted at room temperature under nitrogen

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atmosphere for 5 d in a water/ethanol (1:1, v/v) solvent mixture. After evaporation of the

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ethanol and adjustment of the pH to 7 with 6 N hydrochloric acid, a crude white product

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was obtained by filtration. The product was purified by recrystallization from acetonitrile.

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The purified HA was obtained by filtration and dried at 45 °C overnight.

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HACM copolymer was synthesized by free radical polymerization (Scheme 1). In

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100 mL round-bottom flask an equimolar amount of 3-cloro-2-hydroxypropyl

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methacrylate (CM) (10 mmol) and HA (10 mmol) was dissolved in methanol. After 1 wt

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% azobis(isobutyronitrile) AIBN was added to the flask, nitrogen gas was bubbled

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through the solution for 15 min to remove any dissolved oxygen before initiating the

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reaction. Then the mixture was reacted at 65 °C for 2 h in the presence of AIBN as an

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initiator. The polymerization reaction was performed under reflux conditions under

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nitrogen atmosphere. After completion of the copolymer reaction, the solvent was

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removed by evaporation, and the HACM copolymer was obtained as a white solid in 92

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% yield. The structure of the copolymer was confirmed by FTIR and NMR analyses (see

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Figures 1 and 2).

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Scheme 1. Synthesis of HACM copolymer.

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Antimicrobial Stainless Steel Surface Modification. Stainless steel (SS) sheets

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were cut into 6.45 cm2 coupons. Before surface modification with the synthesized

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copolymer, the stainless steel coupons were subjected to cleaning by soaking first in 6 ACS Paragon Plus Environment

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acetone, then in ethanol, and finally in deionized (DI) water for 10 min in each solvent to

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remove oil and dirt. Cleaned SS coupons were dried in an oven for 1 h at 45 °C prior to

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an oxidation treatment. In order to further clean and create hydroxyl groups on the

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stainless steel surface, the SS coupons were treated by immersion in a piranha solution

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composed of 30 % hydrogen peroxide and sulfuric acid (H2O2: H2SO4 in 7:3 volumetric

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ratio)37 for 30 min and agitated at 120 rpm at 25 °C, and then the coupons were rinsed in

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copious amounts of DI water. The SS coupons were then dried at 130 °C for 10 min and

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kept in sealed poly(propylene) bags. Prior to functionalization with HACM copolymer,

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the SS coupons were first treated in 2 N NaOH for 30 min while agitating at 120 rpm at

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25 °C. The coupons were then rinsed with DI water and dried at 45 °C for 1 h.

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In order to provide antimicrobial properties, the stainless steel coupons were

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subjected to N-halamine chemical modification. Synthesized HACM copolymer was

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dissolved in 3 % (w/v) DMF. Then the SS coupons were immersed in the copolymer

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solution and shaken for 15 min at 25 °C at 120 rpm. Following treatment with the

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HACM copolymer, the coupons were dried under nitrogen atmosphere and then cured at

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130 °C for 1 h to promote covalent bond formation between the oxidized stainless steel

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surface and the HACM copolymer (Scheme 2).

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Scheme 2. N-halamine HACM modification onto stainless steel and subsequent chlorine

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activation.

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Chlorination Process and Analytical Titration of the N-Halamine-modified

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Stainless Steel Surface. The stainless steel coupons, which were treated and

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functionalized with HACM, were subjected to a halogenation process to activate N-H

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sites and form N-Cl bonds on the HACM copolymer to provide antimicrobial properties.

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The SS coupons were chlorinated in a 10 % (v/v) aqueous solution of household bleach

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(8.25 % sodium hypochlorite) at a pH of 7 (adjusted by a 6 N HCl solution) for 30 min.

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After the chlorination process, the samples were first rinsed with tap and then DI water,

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and finally dried at 45 °C for 1 h to remove any occluded free chlorine from the material.

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The amount of oxidative chlorine content (Cl+) of the N-halamine-modified stainless

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steel coupons was determined by standard iodometric/thiosulfate titration.38 The available

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Cl+ atoms/cm2 was calculated using the following equation:

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Total chlorine content (atoms/cm ) =

× 6.02 × 10

(1)

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where N and V are the normality (equiv /L) and volume (L) of the titrant (Na2S2O3),

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respectively, and A is the area of both of the sides of the steel surface used in the titration.

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It was determined that for those stainless steel coupons containing only un-chlorinated

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HACM as controls, the titration procedure yielded no detectable Cl+.

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Stability and Regenerability Testing. The washing stabilities and the re-

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generabilities of the chlorine content of the SS-HACM-Cl coupons were determined. In

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this test 1 v/v% Liqui-Nox solution was prepared in DI water. 50 mL tubes were filled

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with 35 mL of this detergent solution, and the chlorinated coupons were immersed for 45

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min in an orbital shaker at 60 rpm at 25 °C for each cycle. The chlorine contents were

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determined using iodometric titration (eq 1) after each washing cycle and after

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regeneration.

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The UVA light stabilities of the N-halamine-modified SS coupons were also evaluated. A set of HACM coated SS coupons was prepared for the UVA testing. For this 8 ACS Paragon Plus Environment

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testing, the SS coupons were modified with HACM and then chlorinated as described

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above with the chlorine contents determined by iodometric titration after several time

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intervals of UVA irradiation using eq 1.

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Antimicrobial Efficacy Testing. Biocidal efficacies of the HACM-modified SS

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surfaces and the chlorine activated antimicrobial SS surfaces were evaluated against

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Gram-positive S. aureus (ATCC 6538) and Gram-negative E. coli O157:H7 (ATCC

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43895) bacteria. In this procedure25 the bacteria were suspended in 100 µM phosphate

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buffer (pH 7) to produce a suspension of known population (colony forming units, CFU).

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Briefly, each strain of bacteria was streaked onto a trypticase soy agar (TSA) plate and

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incubated at 37 °C for 16 h. Bacterial suspensions were prepared by suspending bacterial

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colonies swabbed from the TSA plates in 100 µM Butterfield's phosphate buffer (BPB).

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Bacterial populations of the bacterial suspensions were estimated by using a microplate

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spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) at O.D.640 nm , and inocula

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with determined populations of about 4x107 CFU/mL were prepared. Then, an aliquot of

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25 µL of this suspension (containing ca. 6 logs of CFU) was placed in the center of a 2.54

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cm x 2.54 cm square coupon (HACM-treated or a native non-treated control), and a

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second identical coupon was placed on top. Both coupons were covered by a sterile

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weight to ensure good contact with the bacteria. After predetermined contact times (5, 10,

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15, 30 and 60 min), the coupons were quenched by 5.0 mL of sterile 0.02 N sodium

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thiosulfate solution to neutralize any remaining oxidative chlorine and thus terminate the

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disinfection action. The quenched samples were first vortexed for 2 min, and then ten-

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fold serial dilutions were prepared using pH 7, 100 µM phosphate buffer. Then the

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dilutions were plated on trypticase soy agar plates. After the plates were incubated at 37

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°C for 24 h, bacterial colonies were counted for the biocidal efficacy analysis. The

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number of bacteria (CFU/sample) of inoculum and number of bacteria at each contact

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time was calculated as shown below:

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CFU/sample = (Number of bacteria colonies counted on agar plate x Dilution

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factor x 5 mL) / Plated volume (0.025 mL)

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(2)

The results were reported as log CFU/sample reduction calculated from the equation 9 ACS Paragon Plus Environment


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below: Log Reduction = log10 (A) - log10 (B)

(3)

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Where A is the population of the bacteria before treatment (control sample) (bacterial

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population of the inoculum obtained by the agar plate count method), and B is the

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population of the bacteria after treatment (bacterial population of the inoculum obtained

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by the agar plate count method at each contact time).

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Two coupons were used in each experiment for each collected datum for each

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type of treatment against one type of bacteria at each determined contact time (5, 10, 15

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and 30 min). As a result, in each experiment 24 coupons were tested against one type of

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bacterium. All experiments were performed at least three times (on different days) using

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fresh bacterial inocula for each type of bacterium.

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RESULTS AND DISCUSSION

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Synthesis and Characterization of the HACM Copolymer. 2-acrylamido-2-

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methyl-1-(5-methylhydantoinyl)propane (HA) precursor (Scheme 1) was synthesized

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according to a protocol described previously23 and analyzed by 1H NMR and FTIR

238

spectra (Supporting information, Figures S2 and S3). In this study precursor HA was

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used because of its ability to potentially store higher chlorine content as a result of three

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N-H sites within its structure. HACM copolymer was synthesized by free radical

241

polymerization in the presence of AIBN catalyst as illustrated in Scheme 1. FTIR and

242

NMR spectra were employed to characterize the synthesized HACM copolymer. The 1H

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NMR spectrum of the synthesized HACM copolymer is shown in Figure 1. The mole

244

fraction of HA in the copolymer was calculated by comparing the signal area of total

245

methylene group protons (g, 3.62 ppm), adjacent to the chlorine atom (-CH2-Cl) of the

246

CM moiety, to the imide proton signal area (m, 10.60 ppm) of the HA moiety.

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Consequently, the reactivity ratio of HA was slightly lower than that for CM, resulting in

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slightly higher CM amount (n) in the copolymers as compared to the feed ratio; m/n was

249

0.95, whereas the feed ratio was 1.00.


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250 251


Figure 1. 1H NMR spectra of the synthesized HACM copolymer (solvent: DMSO-d6).

252

FTIR spectra of the 3-chloro-2-hydroxypropyl methacrylate (CM) and hydantoin

253

acrylamide (HA) precursors and synthesized HACM copolymer are shown in Figure 2. It

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was observed that vinyl bands at 1636 cm-1 and 1621 cm-1 on the CM and HA spectra,

255

respectively, disappeared in the copolymer spectra indicating a complete

256

polymerization.23 Carbonyl stretching bands of the imide and heterocyclic and acyclic

257

amide groups of HACM could clearly be observed at 1767, 1708, and 1656 cm-1,

258

respectively. Vibrational stretching mode bands characteristic of the hydantoin carbonyl

259

moieties of HA and HACM were observed at 1767 and 1708 cm-1.39 In addition the C-N

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vibrational stretching mode band for the acyclic amide group of HA and HACM was

261

observed at 1537 cm-1. The ester carbonyl stretching mode band for CM was identified at

262

1707 cm-1. The band due to C-Cl bond stretching on the CM monomer and the HACM

263

copolymer was observed at 751 cm-1.



100

%T

80 60

A 3444

%T

100

751

1636 1707

40 B

80 1767

60

3206

40 100

%T

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731

1664 1708

C

80 60

1621 1537

1767 3290

1537 1656

1708

40

3500 3000 2500 2000

1500

Wavelength cm

751

1000

-1

264 265

Figure 2. Representative ATR-FTIR spectra of precursor A: 3-cloro-2-hydroxypropyl

266

methacrylate (CM); precursor B: hydantoin acrylamide (HA) and C: synthesized

267

copolymer (HACM).

268

Characterization of HACM Copolymer-modified Stainless Steel (SS).

269

Attachment of the HACM copolymer onto the stainless steel surface was confirmed by

270

ATR-FTIR spectroscopy as shown in Figure 3. Unmodified native stainless steel (SS),

271

HACM modified but unchlorinated (SS-HACM), and HACM-modified and chlorinated

272

stainless steel (SS-HACM-Cl) spectra were observed. As expected the unmodified native

273

SS sample provided no vibrational bands. After chlorination of the HACM-treated

274

stainless steel surface, the N-H bonds were transformed into N-Cl bonds. As a result, the

275

vibrational stretching bands of the hydantoin carbonyl groups (amide and imide) in the

276

1720 to 1780 cm-1 region were shifted to higher wavenumber as observed previously for

277

chlorinated hydantoin moieties.23,28,29 In addition, as seen in the close up FTIR spectra of

278

the 3800-2500 cm-1 region the broad N-H band of the SS-HACM at 3235 cm-1 got

279

weaker and shifted to higher wavelength in the spectrum of SS-HACM-Cl. O-H


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280

vibrational band was detected centered at about 3355 cm-1 which can be attributed to the

281

presence of residual water.

95

90 85 100

%T

A

95 A

B

95 3235

90 85 100 90 80 70

1764

751

1655

1725 C 3355

% Transmission

%T

100

%T

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


90

B C

3355

751

1783 1732

1662

85 4000

3235

2967

3500 3000 -1 Wavelength cm

2500

4000 3500 3000 2500 2000 1500 1000 -1 Wavelength cm

282 283

Figure 3. ATR-FTIR spectra of A: native stainless steel (SS); B: N-halamine HACM

284

modified, but un-chlorinated, stainless steel (SS-HACM); and C: N-halamine HACM

285

modified and chlorinated stainless steel (SS-HACM-Cl).

286

Further surface characterization was effected using XPS. The XPS analyzer was a

287

127 mm radius double-focusing concentric hemispherical energy analyzer (CHA)

288

equipped with an aberration compensated input lens (ACIL). XPS spectra were recorded

289

in the fixed analyzer transmission (FAT) mode with a pass energy of 80 eV, appropriate

290

for acquisition of medium resolution, high signal-to-noise spectra. The magnification of

291

the analyzer in the FAT mode was selected to collect electrons from the smallest

292

allowable (2 mm2) area on the specimen. The resolution of the instrument at the operating

293

parameters was measured from FWHM of the Ag3d5/2 peak to be 1.0 eV. The XPS

294

energy scale was calibrated by setting the Ag3d5/2 line on clean silver to exactly 368.3 eV

295

referenced to the Fermi level. Due to specimen charging during X-ray irradiation, the

296

energy axis of each XPS spectrum has been shifted to make the C1s binding energy line

297

equal to 285.0 eV, which is a standard hydrocarbon energy, (C-H and C-C bonds) used to

298

reference charge affected materials. The potential measured on a typical sample was 1-3

299

V. The photoelectrons were excited by a water-cooled, conventional (ie., non-

300

monochromatic) dual anode X-ray gun equipped with an Al window. The angle of the 13 ACS Paragon Plus Environment


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301

incidence of the X-ray beam with the specimen normal was 51.5o. MgKα (1253.6 eV)

302

radiation was used exclusively. The XPS surface composition was calculated based on

303

the Scofield cross-sectional values accounting for the instrumental transmission function

304

in the FAT mode of operation.40

305

Survey XPS spectra in the 0 – 600 eV binding energy (BE) region for SS, SS-

306

HACM, and SS-HACM-Cl coupons are shown in Figure 4. The BE values to be

307

discussed below were measured from expanded spectra. The two primary bands

308

displayed for SS are at 531.9 and 285.0 eV for O1s and C1s, respectively. The O1s band

309

is the most intense representing 44.2 % of the spectrum as compared to 35.7 % for the

310

C1s band. This can be attributed to the surface oxidation treatment with piranha solution

311

followed by aqueous base creating O- groups on the SS surface. Upon treatment with

312

copolymer HACM to form SS-HACM, the relative contribution of the O1s band

313

decreases to 13.9 % as compared to the C1s band at 69.5 %. The O1s band also shifts to

314

higher BE at 532.1 eV. This observation is consistent with O- groups on the SS surface

315

reacting with C-Cl functionalities on HACM to form covalent bonds in a nucleophillic

316

substitution with Cl- ions as leaving groups. The O- groups, now losing their formal

317

negative charge, would be expected to provide bands at higher O1s BE as observed. The

318

resolution of the XPS was not sufficient to distinguish between the several types of

319

covalently bound oxygen on the copolymer and surface. Also appearing in the XPS

320

spectrum for SS-HACM were prominent bands for nitrogen (N1s at 399.8 eV, 5.3 %) and

321

chlorine (Cl2p at 200.6 eV, 9.1 %), which were not present in the XPS spectrum for SS.

322

The presence of nitrogen is firm evidence that HACM is indeed attached to the surface of

323

SS to form SS-HACM. The presence of chlorine indicates that not all of the C-Cl

324

functionalities in HACM were reacted with O- groups on SS. This form of chlorine is not

325

oxidative (Cl+) and, as such, its presence can not be determined by our titration

326

procedure. Upon oxidative chlorination with dilute bleach to form N-Cl polar covalent

327

bonds in producing SS-HACM-Cl, little change in the XPS spectrum other than an

328

increase in the contribution of the Cl2p band would be expected. This was the case as

329

bands for oxygen (O1s at 532.1 eV, 13.8 %), carbon (C1s at 285.0 eV, 64.0 %), nitrogen

330

(N1s at 399.8 eV, 6.1%), and chlorine (Cl2p at 200.6 eV, 16.1 %) were obtained. The


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331

resolution of the XPS spectra was not sufficient to distinguish slight changes in oxidation

332

states of the elements present, eg. N-H, N-Cl, and C-Cl. The only accurate means to

333

quantitate the amount of oxidative chlorine, the source of antimicrobial activity, is with

334

an iodometric/thiosulfate titration as has been done in this work. In summary, XPS

335

provides evidence consistent with our hypothesis that the copolymers HACM and

336

HACM-Cl are covalently bound to the surface of SS.

337

338 339

Figure 4. XPS of native stainless steel (SS); N-halamine HACM modified, but un-

340

chlorinated, stainless steel (SS-HACM); and N-halamine HACM modified and

341

chlorinated stainless steel (SS-HACM-Cl).

342

The effect of chlorination condition on active chlorine loadings of the HACM

343

modified SS coupons was determined (Table 1). The active chlorine content of the

344

HACM-SS coupons chlorinated for 30 min at different household bleach concentrations

345

(1, 5, and 10 v/v %) was observed. As seen in Table 1, the active chlorine content was 15 ACS Paragon Plus Environment


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346

increased from 5.05 x1016 atoms/cm2 to 1.47 x1017 atoms/cm2 with an increase of the

347

sodium hypochlorite concentration. In the previous study of SS modified with branched

348

poly(ethyleneimine) and poly(acrylic acid), a layer-by-layer deposition enabled the

349

authors to obtain a chlorine loading of about 2.2 x 1016 atoms/cm2. We have noted

350

previously that a chlorine loading of at least 1 x 1016 atoms/cm2 is necessary to obtain

351

antimicrobial efficacy for any surface modified by a chloramine.41

352

Table 1. Chlorine loadings of the HACM modified SS coupons at different

353

concentrations of sodium hypochlorite. Each datum represents an average of three

354

SS coupons (2.54 cm x 2.54 cm in size). SS-HACM-Cl

355

Bleach concentration

Cl+

(wt %)

(atoms/cm2)

1

5.05 x 1016 ± 6.77 x 1015

5

7.73 x 1016 ± 1.13 x 1016

10

1.47 x 1017 ± 4.04 x 1016

In addition, the chlorination reaction times of the modified SS coupons were

356

evaluated. The coupons were chlorinated in 10 v/v % household bleach solution at

357

different time intervals for 15, 30, 45 and 60 min, respectively (Figure 5). The

358

chlorination time did not significantly influence the active chlorine content; only a slight

359

increase of the chlorine loading was observed at extended times. A chlorination time of

360

30 min was chosen the remainder of the study.


Page 16 of 27

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2.000E+17

Cl+ atoms/cm2

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


1.800E+17 1.600E+17 1.400E+17 1.200E+17 1.000E+17 0

20

40

60

80

Time (min) 361 362

Figure 5. Chlorine loadings of the HACM modified SS coupons at different chlorination

363

time intervals. Each datum point represents an average of three SS coupons (2.54 cm x

364

2.54 cm in size).

365

Stability Testing. Washing and re-chargeability of the chlorine activated HACM

366

modified stainless steel samples were evaluated, and the results are illustrated in Table 2.

367

It was found that after 5 washing cycles SS-HACM-Cl samples possessed most of their

368

initial chlorine loadings. Re-chargeability of the chlorine content of the HACM-Cl-

369

modified samples was also measured. The samples were capable of being reactivated to

370

their initial contents after the re-chlorination procedure. These data showed that HACM

371

attachment onto stainless steel is stable, and after exhaustion of the chlorine, the N-Cl

372

bond can be regenerated repeatedly over numerous cycles without the necessity of

373

reapplication of the HACM coating.

374

The stabilities of the un-chlorinated and chlorinated HACM modified stainless

375

steel samples towards UVA light irradiation are presented in Table 3. The oxidative

376

chlorine content (Cl+ %) decreased significantly with the extension of UVA light

377

exposure time. After 1 h of UVA light exposure, SS-HACM-Cl samples lost about 44%

378

of the bound chlorine dropping from 9.19 x 1016 atoms/cm2 to 5.11 x 1016 atoms/cm2.

379

However, after 1 h the number of available chlorine atoms did not significantly change

380

upon continuous UVA exposure. Chlorine content of the samples remained at around 5 to

381

4 x 1016 atoms/cm2 until a 12 h exposure time. After 12 h of UVA light irradiation, 63%

382

of the bound chlorine was lost. A portion of the oxidative chlorine could not be recovered 17 ACS Paragon Plus Environment


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383

after exposing to dilute sodium hypochlorite. This could be due to the intramolecular

384

rearrangement of the chlorine atoms onto carbon atoms of the copolymer followed by

385

decomposition and detachment from the SS surface. This phenomenon has been observed

386

by Worley and coworkers in previous studies of hydantoinyl siloxane polymers attached

387

to cellulose.42 It has been reported that decomposition and the loss of the chlorine moiety

388

of Si-alkyl functional N-halamine-coated cotton substrates due to a scission reaction can

389

be improved by addition of TiO2 moieties into the structure.43 Further studies need to be

390

conducted in order to confirm the mechanism of this decomposition process for HACM

391

on stainless steel.

392

Table 2. Washing stability of HACM copolymer modified stainless steel (SS-HACM-

393

Cl). Washing cycle

X

Y

Cl+ atoms/cm2

Cl+ atoms/cm2

Initial

5.14 x 1016

5.14 x 1016

1

6.42 x 1016

6.13 x 1016

2

4.52 x 1016

7.00 x 1016

3

4.38 x 1016

5.54 x 1016

5

4.52 x 1016

6.85 x 1016

394

X: HACM modified SS coupons were chlorinated before washing and titrated after

395

washing; Y: HACM modified SS coupons were chlorinated before washing and re-

396

chlorinated after washing and titrated. Each datum represents an average of three SS

397

coupons (2.54 cm x 2.54 cm in size).

398 399

Table 3. UVA light stability of activated chlorine on the HACM modified stainless

400

steel coupons. Exposure Time

X

Y

(h)

Cl+ atoms/cm2

Cl+ atoms/cm2

0

9.19 x 1016

1

5.11 x 1016

2

4.96 x 1016

5.83 x 1016


Page 18 of 27

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3

5.98 x 1016

6

4.38 x 1016

4.96 x 1016

12

3.35 x 1016

4.38 x 1016

401

X: HACM-modified SS coupons were chlorinated before UVA exposure, the chlorine

402

remaining determined for the coupons after UVA exposure; Y: HACM-modified and

403

chlorinated SS coupons were re-chlorinated after UVA exposure. Each datum represents

404

an average of three SS coupons (2.54 cm x 2.54 cm size).

405

Antimicrobial Efficacy. The antimicrobial efficacy results for the native non-

406

modified SS, modified un-chlorinated HACM, and modified and chlorinated HACM-Cl

407

stainless steel samples have been summarized in Table 4. Average available chlorine

408

contents of the halogen-activated SS-HACM-Cl coupons, used for antimicrobial efficacy

409

analysis, were measured as 9.6 x 1016 ± 1.9 x 1016 atoms/cm2. All samples (control, SS-

410

HACM and SS-HACM-Cl) were challenged with S. aureus and E. coli O157:H7 at

411

approximately 6 logs (CFU/sample) of initial inoculum concentration for both S. aureus

412

and E. coli O157:H7.

413

As expected, control native stainless steel coupons and HACM modified stainless

414

steel (SS-HACM) coupons showed a limited degree of bacterial reduction. These results

415

are in agreement with those for N-halamine-modified textile surfaces for which some

416

viable CFU’s could not be recovered using the experimental procedure. The control

417

native stainless steel coupons and HACM modified stainless steel (SS-HACM) coupons

418

did not inactivate any bacteria; the variability in the data in Table 4 for these control

419

coupons and for the coupons of SS-HACM-Cl for which complete inactivation was not

420

obtained can be attributed to the challenging microbiology analysis procedure. The SS-

421

HACM-Cl stainless steel coupons provided a complete inactivation of about 6 logs (the

422

limit of detection for the antimicrobial assay) against S. aureus and E. coli O157:H7

423

bacteria within 15 min of contact time in all three replicate assays. To the best of our

424

knowledge, these results show superior inactivation time and efficacy against both Gram-

425

positive and Gram-negative bacteria when compared to previous antimicrobial modified

426

stainless steel surfaces. Probably these observations are a result of higher chlorine

427

loadings for the HACM-Cl on SS than was achieved in the study of SS modified by a 19 ACS Paragon Plus Environment


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428

chlorinated copolymer of poly(ethyleneimine) and poly(acrylic acid).37

429

Table 4. Antimicrobial efficacy of control-SS (native non-modified), SS-HACM

430

(HACM modified and un-chlorinated), SS-HACM-Cl (HACM modified and

431

chlorinated) stainless steel coupons against E.coli 0157:H7 and S. aureus. Samples

Contact time (min)

Inoculum

Control-SS HACM-SS HACM-SS-Cl

60 60 5 10 15 30 60

Bacterial log reduction E.coli O157:H7 Exp1 Exp 2

Exp3

6.20

5.99

6.30

0.45 2.34 3.12 ND* 6.20 6.20 6.20

0.46 2.28 4.16 ND* 5.99 5.99 5.99

1.03 1.05 0.75 1.79 6.30 6.30 6.30

432 Samples

Contact time (min)

Inoculum

Bacterial log reduction S. aureus Exp1

Exp2

Exp3

6.24

5.85

5.83

Control-SS HACM-SS HACM-SS-Cl

433 434 435 436 437 438 439 440 441

60 1.46 1.34 1.27 60 2.21 2.16 1.32 5 3.71 3.72 0.96 * * 10 ND ND 5.83 15 6.24 5.85 5.83 30 6.24 5.85 5.83 60 6.24 5.85 5.83 1 Exp 1: The inoculum concentrations were 6.20, and 6.24 logs for E. coli O157:H7 and S. aureus, respectively. 2 Exp 2: The inoculum concentrations were 5.99, and 5.85 logs for E. coli O157:H7 and S. aureus, respectively. 3 Exp 3: The inoculum concentrations were 6.30, and 5.83 logs for E. coli O157:H7 and S. aureus, respectively. * ND: No determination.

FUTURE STUDIES 20 ACS Paragon Plus Environment

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442


Before the antimicrobial stainless steel modified with the newly synthesized N-

443

halamine coating discussed in this work can enter practical application, long-term

444

stability testing under hydrothermal conditions as well as toxicity/cytotoxicity and

445

biocompatibility data will need to be evaluated.44 It is encouraging that there are reports

446

of N-halamine polymer coatings on other surfaces which have indicated that toxicity

447

problems are not likely to be an issue for the N-halamine-treated stainless steel. 45-47

448

CONCLUSIONS

449

A new hydantoin copolymer, containing N-halamine forming moieties, was

450

synthesized and chlorinated in order provide antimicrobial stainless steel coatings. The

451

work demonstrated a successful stainless steel surface modification through covalent

452

attachment of HACM copolymer via grafting onto the surfaces. The stainless steel

453

surfaces provided excellent antimicrobial properties against S. aureus and E. coli

454

O157:H7 bacteria upon activation of the N-halamine sites by a chlorination process.

455

Compared to previous reported research, this newly developed antimicrobial N-halamine

456

copolymer-modified stainless steel exhibited superior antimicrobial properties and

457

deactivated 6 logs of Gram-negative and Gram-positive bacteria populations within only

458

15 min of contact time. Washing results showed excellent stability and durability of the

459

HACM-Cl stainless steel coating. In addition, after 5 cycles of washing, the test samples

460

could be recharged to their initial chlorine contents suggesting that a strong and stable

461

copolymer attachment onto the surface could be achieved. However, UVA irradiation

462

caused moderate decomposition of the coatings; after 12 h of UVA exposure time, a

463

sufficient amount of chlorine remained for bacterial inactivation, and the samples could

464

be partially re-chlorinated. Further work is ongoing to explore means of stabilizing the

465

coatings against photolytic decomposition. The antimicrobial stainless steel surfaces

466

developed in this study possesses reasonable potential for use in industries (such as food

467

and biomedical) in which cross contamination and biofilm formation is a continuous

468

problem.

469

ASSOCIATED CONTENT

470

Supporting Information 21 ACS Paragon Plus Environment


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471

Proof of successful synthesis of monomer HA. This material is available free of charge

472

online at http://pubs.acs.org.

473

AUTHOR INFORMATION

474

Corresponding Author

475

*

476

Notes

477

The authors claim no competing financial interest.

478

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480

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