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Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces Chanchan Wang, Dominic Sauvageau, and Anastasia L. Elias ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08664 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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ACS Applied Materials & Interfaces

Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces Chanchan Wang, Dominic Sauvageau*, Anastasia Elias* 9107 - 116 Street, Department of Chemical and Material Engineering, University of Alberta, Edmonton, T6G 2V4, Canada First author: [email protected] Corresponding authors: [email protected]; [email protected] Abstract A rapid, efficient technique for the attachment of bacteriophages (phages) onto polyhydroxyalkanoate (PHA) surfaces has been developed and compared to three reported methods for phage immobilization. Polymer surfaces were modified to facilitate phage attachment using: (1) plasma treatment alone, (2) plasma treatment followed by activation by 1ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride

(EDC)

and

N-

hydroxysulfosuccinimide (sulfo-NHS), (3) plasma-initiated acrylic acid grafting, or (4) plasmainitiated acrylic acid grafting with activation by EDC and sulfo-NHS. The impact of each method on the surface chemistry of PHA was investigated using contact angle analysis and X-ray photoelectron spectroscopy. Each of the four treatments was shown to result in both increased hydrophilicity and in the modification of the surface functional groups. Modified surfaces were immersed in suspensions of phage T4 for immobilization. The highest level of phage binding was observed for the surfaces modified by plasma treatment alone. The change in chemical bond states observed for surfaces that underwent plasma treatment is suspected to be the cause of the increased binding of active phages. Plasma-treated surfaces were further analyzed through phage-staining and fluorescence microscopy to assess the surface density of immobilized phages

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and their capacity for host capture. The infective capability of attached phages was confirmed by exposing the phage-immobilized surfaces to the host bacteria Escherichia coli in both plaque and infection dynamic assays. Plasma-treated surfaces with immobilized phages displayed higher infectivity than surfaces treated with other methods; in fact, the equivalent initial multiplicity of infection was two orders of magnitude greater than with other methods. Control samples – prepared by immersing polymer surfaces in phage suspensions (without prior plasma treatment) – did not show any bacterial growth inhibition, suggesting they did not bind phages from the suspension. Keywords: Bacteriophage immobilization; surface modification; phage binding; inhibition of E. coli; detection of bacteria, active material. Introduction Increasing public health concerns related to bacteria contamination of food, beverage, medication and cosmetics have prompted interest in the development of low-cost, specific and efficient pathogen capture, detection and treatment materials. The conventional detection methods rely on microbiological techniques, which require sophisticated procedures, restricted growing condition, long time and well trained personnel. The detect limit is also relatively high. Bioactive polymers resulting from the conjugation of polymeric materials with biomolecules or microorganisms can be used for a variety of detection or treatment application, including biomedical applications

1

and pathogen sensing

2, 3

, which relies on the range of properties

displayed by the various biocompatible polymers that may be employed, and most importantly the recognition moieties. Bacteriophages (or phages) – viruses that attack bacteria – can be used as specific recognition moieties in developing bioactive polymers for pathogen capture, detection or 2 ACS Paragon Plus Environment

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treatment

4, 5

. Lytic phages selectively capture bacteria via binding of the tail fibers to specific

bacterial receptors; they then infect and replicate within the bacteria, eventually lysing the host and releasing more phages. Phages are generally stable, cheap to produce, commonly found in the environment and food products, and have low immunogenicity 6. Phages display a greater specificity compared to other antimicrobial agents (e.g. silver nanoparticles, antimicrobial peptides, polymers, chemicals, etc.). These characteristics of phages are beneficial in applications such as active packaging 4, bacterial infection diagnostics 7, 8 and biofilm treatment 9. In pathogen detection applications, phages can be used as a recognition element to selectively capture desired types of bacteria with high specificity. They have been integrated into a variety of sensing/detecting platforms, including those based on optical detection methods 10, microbead assays

11

, mass-based techniques (including quartz crystal microbalances

12

), surface plasmon

resonance 13, cantilevers 14, and electrochemical sensing 15, 16. Techniques have been developed for binding phages on gold 17, 18, cellulose 4, and silica surfaces

19

. Gold surfaces have been modified with sugars (dextrose and sucrose) as well as

amino acids (histidine and cysteine) to mediate phage attachment 17. Physical adsorption has also been used successfully to immobilize filamentous phages on gold surfaces to detect Escherichia coli and Listeria monocytogenes 4, 13. For cellulose, positively charged chemicals have been used to modify the surface and bind bacteriophages electrochemically 4. In the case of silica particles, diverse surface modification agents have been used to enable phage immobilization 19. Covalent bonding to polymers has been performed using plasma polymerization to introduce carboxyl groups on the polymer surface, followed by crosslinking of these groups to the amines in the proteins on the surface of the phages

20

. In this method, 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride (EDC) was used to activate the crosslinking reaction, and N-



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hydroxysulfosuccinimide (sulfo-NHS) to improve the reaction efficiency. This covalent bonding method produces surfaces with good phage activity and high surface density; however, the chemical reagents utilized in this multi-step procedure introduce extra concerns with regards to biocompatibility. For example, EDC is known to be toxic to some cell types, irritates the skin and may cause severe eye damage 21.

For all of the cases described above, there exist several challenges to using bacteriophages as bioconjugates. These include retaining infectivity after immobilization, achieving a high density of active phages on surfaces, controlling the orientation of phage particles (to ensure tail fibers are exposed to the host, which requires an orientation normal to the plane of the surface) and designing a scalable process

22

. Finally, to the best of our knowledge,

no studies have examined the conjugation of phages directly with plasma-treated polymer surfaces. Plasma treatment has been used to facilitate the attachment of polymers23,24 and biological elements25-28 to a variety of surfaces. In terms of biological elements, plasma treatment of the surface has been used to bind albumin and human mammary epithelial cells to plasma-treated polystyrene

25

, nisin to a plasma-treated polyester fabric

26

, L929 cells (mouse

fibroblast-like cell line) to argon- and nitrogen-plasma modified chitosan membranes lysozymes to plasma-treated polyethylene films (for enhanced antimicrobial activity)

28

27

, and

. In all

these studies, the adsorption of the biological elements improved with the increased hydrophilicity of the polymer surfaces resulting from the plasma treatment. Our work focuses on the attachment of phages to polyhydroxyalkanoate (PHA), a biopolymer that is produced by bacteria. This material is of particular interest for packaging applications due to the fact that it is


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food compatible and biodegradable 29. A bioactive material formed from phage-conjugated with PHA could thus have application as an antimicrobial packaging material. The present study describes a simplified approach to immobilizing phages on a PHA surface through the use of an oxygen-plasma treatment, and compares its efficiency to diverse phage attachment methods. 2. Materials and Methods

2.1 Chemicals and Cell Cultures Polyhydroxyalkanoate (PHA) pellets, made of 99% natural PHA, were purchased from Bulk Reef Supply Inc. (Golden Valley, MN, USA). Chemical analysis (by CHNS elemental analysis) has shown that this material contains ~1% Si (as determined by XPS), and that the C:O and C:H ratios are consistent with high molecular weight polyhydroxybutyrate (PHB, a type of PHA 30). EDC (≥98%) and sulfo-NHS (98%) were purchased from Sigma-Aldrich (Mississauga, Canada) and used as received. Acetic acid (glacial) was purchased from Fisher Scientific (Ottawa, Canada) and was used as received. Agar (DifcoTM) and Typtic Soy Broth (TSB, Bacto) were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). 2-(Nmorpholino)ethane sulfonic acid (MES, ≥99%) was purchased from Sigma-Aldrich (Mississauga, Canada). Bacterial cultures of Escherichia coli ATCC 11303 were grown overnight in 25 ml of TSB incubated at 37 °C and 200 rpm in an incubator-shaker (Ecotron, Infors HT, Montreal, Canada) to a concentration of 109 colony forming units per ml (cfu/ml). A phage stock of phage T4 (ATCC 11303-B4), at a titer of 3 x 1010 plaque forming units per ml (pfu/ml), was used in all experiments – as is or diluted – and stored at 4 oC in TSB.



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2.2 Casting of PHA films To prepare a film, 1 g of PHA pellets was dissolved in 40 ml of acetic acid. The mixture was brought to 140 °C on a hotplate-stirrer (Fisher Scientific, Ottawa, Canada) and mixed until the polymer was completely dissolved. 5 ml of prepared PHA solution was then dropped on a glass slide with dimensions 35 mm × 75 mm. The glass slide was put on top of the hotplate and cured for 10 min at 100 °C. After curing, the glass slide was immersed and rinsed multiple times in deionized water and let to sit for 24 h. The film was then vacuum dried to ensure complete removal of the acetic acid. A typical film was 300 µm thick.

2.3 Surface Modification by Plasma Treatment In this study, PHA polymer film squares of 2 cm × 2 cm were treated with oxygen plasma. Plasma treatment was performed through Reactive Ion Etching (µEtch) in a cylindrical chamber 30 cm in diameter. A uniform glow discharge was created within a copper coil wrapped onto the chamber and connected to a radiofrequency (RF) generator. Commercially available oxygen gas (99.993%, Praxair, Edmonton, Canada) was input into the chamber. Samples of PHA polymer films were treated in plasma at a floating potential for 1 min under a RF power of 100 W, an oxygen concentration of 25% and a vacuum of 100 mTorr.

2.4 Water Contact Angle

Contact angle measurements were performed to investigate the hydrophilicity of the surfaces before and after oxygen plasma treatment (applied to each surface of the film). Measurements were performed using the sessile drop method on a Video Contact Angle 2000 System (AST Products Inc., Billerica, MA, USA) with deionized water. For each condition, 3


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samples were prepared, and 3 drops were measured on each surface of the film. Measurements were recorded 30 seconds after the liquid was contacted with the surface.

2.5 X-ray Photoelectron Spectroscopy (XPS)

XPS measurements were performed in order to characterize the surface composition of PHA films before and after treatments, and the surface changes after phage attachment. The experiments were carried out using a Kratos Axis-Ultra (Kratos Analytical Inc., Manchester, UK) with a monochromatic Al X-ray source. The X-rays energy was 1486.6 eV and the base pressure was approximately 2×10−8 Pa. Triplicate samples were prepared and results were collected from 5 different points on the surface of PHA samples.

2.6 Phage amplification The amplification of T4 phages was achieved by incubating 100 µl of 106 pfu of phages in 5 ml of fresh log-phase E. coli culture for 15 min at room temperature. The mixture was then added to 250 ml of TSB media and was incubated at 37 °C in an incubator-shaker (Ecotron, Infors HT, Montreal, Canada) for 6 h. The media was then filtered through a 0.22-µm filter (EMD Millipore, Etobicoke, Ontario, Canada) to remove any remaining bacterial cells or debris from the media, and centrifuged (Sorvall RC 6 Plus, Thermo Electron Corporation, Waltham, USA) at 42018 rcf for 1.5 h to pellet the bacteriophages from the filtered supernatant. The phage pellet was resuspended in 1.5 ml of PBS buffer and incubated overnight at 4 °C. A phage count was performed using a modified version of the soft agar overlay technique

31

and reported in

pfu/ml. The phage titer was adjusted to 1010 pfu/ml by dilution in PBS buffer prior to the phage immobilization work.



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2.7 Phage Immobilization on PHA Film

2.7.1. Adsorption on Untreated and Plasma Treated (PT) Films Attachment methods were performed in PBS buffer at pH 7.5. Oxygen plasma-treated and untreated PHA films of dimensions 2 cm × 2 cm were immersed in 125-ml shake flasks containing 20 ml of a T4 phage suspension (2×108 pfu/ml) and mixed at 4 °C and 60 rpm for 2 h. After exposure to phages, the films were washed seven times in PBS buffer to remove any unbound or loosely attached phages. After this amount of washes, less than 103 pfu/ml of nonimmobilized phages were detected in the solution. All washing steps were performed at room temperature.

2.7.2. Plasma Treatment Combined with EDC/Sulfo-NHS Activation (PT +EDC+NHS) In this case, phages were covalently bonded, via the primary amines of their surface proteins, to carboxyl groups on the surface of plasma-treated films using EDC and NHS chemistry – a method that has been shown to lead to efficient phage immobilization on mica and glass surfaces

32, 33

. The main drawback of this method is that random orientation of phages on

the surface is achieved 22. The method used in our work was adapted from Pearson et al. 20, and is summarized here. The pH was controlled at pH 6.5 using MES. 2 × 10-3 mol/L of EDC and 5 × 10-3 mol/L of sulfo-NHS were added to 20 ml of MES buffer solution. Four plasma-treated PHA films (20 x 20 x 0.3 mm) were added to this solution within 2 h of exposure to the plasma. The carboxylate groups generated on PHA surfaces from the plasma treatment reacted with EDC and sulfo-NHS to generate sulfo-NHS activated intermediates. Amine-containing residues on the surface of the phage couple with sulfo-NHS to form amide bond linkages

34

. The reaction was

performed in an incubator for 2 hours at 30°C. The films were then immersed for 12 h at 4 °C in 8 ACS Paragon Plus Environment

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20 ml of PBS buffer solution containing 108 pfu/ml of phages. Washing steps were performed as described above.

2.7.3. Plasma-Initiated Acrylic Acid Grafting (PT+AA) Plasma initiated acrylic acid grafting was performed to obtain –COOH terminated PHA surfaces. The grafting method followed Ingaki's polymerization method

35

, and phage

immobilization was achieved following Pearson's methods with some modifications 20. A sealed 100-ml container containing 40 ml of an acrylic acid aqueous solution (10 wt%) was prepared and nitrogen gas was added for 20 min to purge the reaction vessel of oxygen. Plasma treatment was conducted as described above, and the films were transferred into the solution within 10 min. The polymerization reaction was maintained at 30 °C for 2 h. The resulting poly(acrylic acid) (PAA) grafted PHA films were immersed in water overnight and then washed with a large amount of water and ethanol, in sequence, to remove any unreacted acrylic acid monomer and PAA homopolymer. The PAA-grafted PHA films were vacuum-dried in a desiccator at room temperature. The films were then immersed for 12 h at 4 °C in 20 ml of PBS buffer solution containing 108 pfu/ml of phages. Washing steps were performed as described above.

2.7.4.

Plasma-Initiated

PAA-Grafting

Combined

with

EDC/sulfo-NHS

Crosslinking

(PT+AA+EDC+NHS）

This method combined the PAA-grafting method and the EDC/sulfo-NHS crosslinking method. The EDC/sulfo-NHS method was performed on a PHA surface that previously underwent the PAA-grafting protocol. All procedures were followed as described above.

2.8 Fluorescent Microscopy for Imaging Immobilized Phages and Host Capture 9 ACS Paragon Plus Environment


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Fluorescent microscopy was used to visualize the surface density of immobilized phages on the PHA film surfaces. In these experiments, the phages were immobilized on the films (1 cm × 1 cm) and then dehydrated by successively immersing the samples in solutions of 25%, 50%, 80% and 100% ethanol. 10 µl of a diluted solution of SYBR green I stain (Life Technologies, Carlsbad, USA) was dropped on top of each film. The staining process was carried out at room temperature and shielded from light for 30 min. The stained film was rinsed with PBS buffer to remove excess stain and then placed on a glass slide. A drop of anti-fading agent was added before placing the cover slide. Microscopy imaging was performed using a Leica Epifluorescent Microscope (CTR MIC, Wetzlar, Germany) under blue light (exciter filter BP 490 nm). The images were captured using a digital camera (QImaging Retiga EX, Surrey, Canada). The phage density on the surface was calculated using ImageJ. Triplicate samples were used for counting, and 8 images were obtained at different locations on each film. The density (phages/area) was calculated for each image; average values and standard deviations were then calculated. A modification of this method was used to assess the capture of E. coli host cells by the immobilized phages. Three-day old stationary phase E. coli cultures grown in TSB were used to avoid rapid lysis by the phage

36

. Cells were precipitated from 1 ml samples by centrifugation

(Eppendorf Centrifuge 5424 R, Mississauga, Canada) at 4000 × g and 4 °C for 4 min. The TSB supernatant was removed and the cell pellet was resuspended in 1 ml PBS buffer. A second centrifugation/resuspension step was performed to remove any residual TSB. 10 µl of SYBR green I stain diluted solution was used to stain the E. coli cells. The staining process was carried at room temperature and shielded from light for 30 min. The stained cells in PBS buffer were centrifuged. The supernatant with excess stain was discarded and the cells were resuspended in PBS buffer. This washing step was repeated prior to contact with the phage-immobilized film (1 10 ACS Paragon Plus Environment

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cm × 1 cm). The film was soaked for 25 min in a 100 µl suspension containing stained E. coli cells. The time period was chosen to maximize host cell capture while avoiding possible cell lysis. The film was rinsed with PBS buffer to remove any non-captured E. coli cells. It was then placed on a glass slide for microscopy, as described above.

2.9 Plaque Assay for Immobilized Phages on PHA Film Modified plaque assays were used for rapid determination of the presence of active phages on PHA surfaces with immobilized phages. In these experiments, overnight cultures of the bacteria E. coli (with optical density at a wavelength of 600 nm (OD600) of approximately 6) were used. 100 µl of overnight culture was added to 3 ml of TSB soft agar (0.75%) kept at 50 oC and poured onto TSB-agar (1.5%) plates. Once the agar had solidified, casted PHA films treated with the different methods – PT, PT+EDC+NHS, PT+AA, or PT+AA+EDC+NHS – and then conjugated with phages were gently placed on top of the plates. The plates were incubated at 37 °C for 24 h and active phages were observed by the formation of clear lysis plaques forming

under and around the edges of the film. Images were taken to record the plaques produced by the active phages on the film surface. The buffer in which the films were suspended was also tested to observe any free bacteriophages present. Untreated films were used as a control.

2.10 Activity of Phages Immobilized on PHA Films The efficacy of a polymer surface conjugated with phages is dependent on the surface density of the phages, their structural integrity and their orientation. To obtain a comparative measurement of efficacy of the different treatments used, infection dynamics studies were performed. A phage stock at a titer of 2×108 pfu/ml was used to immobilize phages on the film surfaces for the different surface activation methods. The phage-immobilized PHA films were 11 ACS Paragon Plus Environment


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immersed in E. coli cultures, and the OD600 was monitored over the infection period to generate E. coli growth/infection curves. For comparison, similar curves were generated using E. coli cells alone, and E. coli cells infected with known concentrations of free phages. Based on the initial multiplicity of infection (MOI) of these controlled infections, an equivalent initial MOI could be established for each film. Films with greater equivalent initial MOI therefore had greater efficacy. Overnight cultures of E. coli were diluted 100-fold in TSB to an initial OD600 of 0.06. The diluted cultures were incubated at 37 °C for 1-2 h until the OD600 reached a value of 0.1. 20 ml of a diluted E. coli culture was then transferred to a shake flask in which the films were immersed. Infections initiated with suspended phage stocks at initial MOIs of 0.001 and 0.1 were included as positive controls. Pure E. coli cultures, E. coli cultures in which treated films that were not exposed to phages were suspended, and E. coli cultures in which untreated films exposed to phages were suspended were used as negative controls. All infection experiments were performed at 37 °C for 6 h and the OD600 was measured every hour.

3. Results and Discussion

3.1 Characterization of Plasma-Treated PHA Surfaces Figure 1 shows an example of a survey XPS C1 spectrum for a control PHA film and a PHA sample treated with oxygen plasma. The software CasaXPS was used to calculate the area of the peaks obtained for each element. Table S2 showing the elemental composition of PHA surfaces after each treatment can be found in the Supporting Information. After plasma treatment, the magnitude of the C-C, C-H peak at 284.8 eV had decreased with respect to the magnitude of peaks at higher binding energies, indicating that the surface was in a higher


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oxidation state. Incorporation of oxygen to the polymer surface resulted in the formation of different functional groups, such as carboxyl, hydroxyl and peroxide groups, as shown in Figure 1 and Table 1 – this was previously discussed in details by Siow et al. 37. The surface of the ascasted PHA samples had an O/C ratio of 0.42. After treatment with oxygen plasma, the O/C ratio increased to 0.6, which indicates more oxygen was found at the surface. The increased content of oxygen-containing polar groups on the plasma treated surfaces can explain the increased wettability after plasma exposure (Figure 2). Therefore, treatment with oxygen plasma resulted in hydrophilic surfaces with contact angle of approximately 14.96° (Figure 2) compared to the native hydrophobic surface which had a contact angle of 96.83°. Plasma treatment provides good control over the plasma chemistry and the possibility of using highly energetic species in surface modification processes. Carrying out further reactions with other monomers or chemicals can lead to uniform surfaces functionalized with various chemical groups. XPS measurements, which collect data from the outmost 10 nm of a surface, can provide valuable information on the chemical composition of the untreated and treated surfaces. Figure 3 shows the O1s peaks of a control PHA film and plasma-treated PHA samples after treatment with the different surface functionalization methods. The O1s envelope of the samples was decomposed into three components: a peak at 528.0 eV corresponding to C-O-H bonds, a peak at 529.0 eV corresponding to the oxygen element on the double bond of O-C=O* groups, and a peak at 530.5 eV, which can be attributed to the oxygen element on the single bond of O*-C=O groups. Based on these peaks, the surface concentration of the different chemical bonds can be calculated (Table 2). Mainly, the O-C=O*/O*-C=O ratio decreased after plasma treatment, from 1.1 to 0.94 (while the ratio of the amplitudes of the O-C=O*/O*-C=O peaks appears to be increasing, the ratio of the areas under the curves decreases). This indicates that



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oxygen was inserted into the polymer backbone between carbons 38. The O-C=O*/O*-C=O ratio was further decreased to 0.91 and 0.85 after EDC/sulfo-NHS activation for PT+EDC+NHS and PT+AA+EDC+NHS, which suggests that the EDC and NHS reacted with the carboxyl groups of the polymer surface. Moreover, a small additional peak is seen at 533.0 eV for the PT+AA+EDC+NHS sample. This peak can be attributed to N-O groups 39 and shows evidence of the sulfo-NHS ester intermediate produced by EDC/sulfo-NHS activation. 3.2 Characterization of Surfaces after Phage Immobilization Control PHA films and plasma-treated PHA surfaces functionalized using different methods were incubated in phage suspensions to allow for immobilization. Preliminary results have shown a period of 12 h allowed for sufficient and complete phage adsorption for all methods tested. After incubation of untreated films in a phage suspension, XPS analysis revealed only O1s and C1s peaks (see Supporting Information Figure S1) which shows no evidence of phage immobilization. In contrast, for all phage-exposed plasma treated samples, new peaks were observed in the spectra: one due to nitrogen (at 397 eV) and the others due to sodium chloride (Na1s at 1072 eV and Cl2s at 272 eV). These peaks are attributed to the adsorbed phages as NaCl is the essential contained of phage capsid

40

. As stated, similar results were

observed for all phage-exposed samples treated with plasma, with or without another treatment. The complete spectrum of PT+phage is shown in Figure 4 (a). In order to compare the efficacy of different treatment methods on attaching phages to PHA surfaces, verification of chemical groups in the XPS O1s spectra were investigated (Figure 4). The spectrum of the control film without plasma treatment (Figure 4b) was decomposed into three peaks of C-O-H (528.0 eV), O-C=O* (529.0 eV) and O*-C=O (530.5 eV). The spectra of the films treated with the other methods were decomposed into four peaks – with the additional


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peak at 532.5 eV assigned to H2O 41. This water peak is believed to relate to the water molecules trapped in the phage capsid: the double-stranded DNA of phage T4 is tightly coiled inside the capsid, and in an aqueous environment, the osmotic pressure on either side of the capsid will drive water in. This has been shown to cause a slight expansion of the capsid 42. When T4 phages are bound to PHA surfaces, XPS analysis thus shows the corresponding water peak, as can be seen at 532.5 eV in Figure 4 (c-f). For the sake of comparison, the water peak intensity from the O1s spectra of PHA films treated with different methods was used to provide a relative measure of phage density on film surfaces. The ratios of the water peak area to the area of all peaks (O1s) found in Figure (c-f) were 11.28%, 3.84%, 2.27%, 7.66% for PT+phage, PT+EDC+NHS+phage, PT+AA+phage, and PT+AA+EDC+NHS+phage samples, respectively (Table 2). Though the EDC/sulfo-NHS activation contributed to the covalent binding of phages

20

, the results indicate that plasma treated sample (PT+phage) had the most

efficient immobilization with the water peak results suggesting as much as three-times more phages attached. Acrylic acid polymerization – which introduced carboxyl groups to the PHA surface – with and without EDC/sulfo-NHS activation, led to improved immobilization compared to simple EDC/sulfo-NHS activation. Yet the resulting immobilization still corresponded to only 68% of the phage density obtained through plasma treatment alone, as measured by the relative intensity of the water peak. Previous studies have shown improved cell and protein attachment on surfaces using oxygen plasma treatment

25, 26, 35

. Enhanced cell adhesion did not always correlate with the

amount of oxygen present at the polymer surface, but in some cases, such as for mammalian epithelial cells 25, surfaces with higher oxygen content and better wettability showed better cell proliferation. Different mechanisms have been shown to be involved in adsorption of proteins



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and cells on hydrophilic surfaces; however, the role of surface wettability in the overall adsorption process is still not fully understood 43. The interactions between protein and surfaces may also lead to protein conformational changes. It was reported that, in the case of hydrophilic surfaces, many proteins are more likely to maintain their conformation, while in the case of hydrophobic surfaces, they will denature

25

. This phenomenon is protein-specific as it will

depend on the nature, hydrophobicity, stability and structure of the protein itself. In the present study, the chemical groups resulting from oxygen plasma treatment, such as peroxide groups, carboxyl groups, and hydroxyl groups, have improved phage attachment. The specific mechanisms involved still need to be investigated further. 3.3 Plaque Assay Improved immobilization of phage particles on a surface does not necessarily translate into better bacterial recognition or better antibacterial capacity. This is mostly due to the fact that to capture bacteria, bound phage particles must retain an active conformation (integrity of detection and/or infection organelles) and be oriented in the proper direction (bound phages have a tendency to lay flat on surfaces upon attachment and drying, and lose their activity 22). Thus plaque assays were performed using the different polymer films prepared with immobilized phage T4 to assess the effectiveness of bacterial recognition and bacterial infection. Control PHA surfaces (which were not subjected to any treatment but were soaked in a phage stock and rinsed), did not display significant lysis (Figure 5 a)). In contrast, PHA surfaces with attached T4 phages, regardless of the method used, led to noticeable lysis under and around the material, as manifested by the clear zone observed around each film (Figure 5 b-e)). This clearly shows that a significant portion of the phages retained their capacity to bind and infect their host after immobilization.


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In order to verify that phages were strongly bound to the surface using the oxygen plasma treatment method, samples were sonicated to try to remove the phages from the surface (Ultrasonic Bath, 40 KHz, Fisher Scientific, PA, USA). Samples were sonicated for 20 min at room temperature in PBS buffer. Figure 6 shows the plate assay results for samples prior to and after sonication, and for the titer of the supernatant after sonication. Minimal changes in the plaque are seen for the films. This suggest that the phages are stable on the surface before and after sonication. The small amount of phage particles detected in the supernantant postsonication were considered as loosely bound phages and accounted for

Immobilization of Active Bacteriophages on ... - ACS Publications

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