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Marine bio-inspired underwater contact adhesion Sean K. Clancy, Antonio Sodano, Dylan J Cunningham, Sharon Sherry Huang, Piotr J. Zalicki, Seunghan Shin, and B. Kollbe Ahn Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00300 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 5, 2016
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Biomacromolecules
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Marine bio-inspired underwater contact adhesion
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Sean K. Clancy§†, Antonio Sodano §†, Dylan J. Cunningham§†, Sharon S. Huang§†, Piotr J.
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Zalicki§†, Seunghan Shin‡*, and B. Kollbe Ahn†*
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†
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USA
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‡
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Chungnam 31056, South Korea
Marine Science Institute, University of California Santa Barbara, Santa Barbara, CA 93106,
Green Materials and Process Group, Korea Institute of Industrial Technology, Cheonan,
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ABSTRACT: Marine mussels and barnacles are sessile biofouling organisms that adhere to a
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number of surfaces in underwater environments and maintain remarkably strong bonds. Previous
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synthetic approaches to mimic biological wet adhesive properties have focused mainly on the
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catechol moiety, present in mussel foot proteins (mfps), and especially rich in the interfacial
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mfps, e.g., mfp-3 and -5, found at the interface between the mussel plaque and substrate.
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Barnacles, however, do not use Dopa for their wet adhesion, but are instead rich in non-
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catecholic aromatic residues. Due to this anomaly, we were intrigued to study the initial contact
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adhesion properties of copolymerized acrylate films containing the key functionalities of
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barnacle cement proteins and interfacial mfps, e.g., aromatic (catecholic or non-catecholic),
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cationic, anionic, and/or non-polar residues. The initial wet contact adhesion of the copolymers
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was measured using a probe tack testing apparatus with a flat-punch contact geometry. The wet
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contact adhesion of an optimized, bio-inspired copolymer film was ~15.0 N/cm2 in deionized
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water and ~9.0 N/cm2 in artificial seawater, up to 150 times greater than commercial pressure-
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sensitive adhesive (PSA) tapes (~0.10 N/cm2). Furthermore, maximum wet contact adhesion was
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obtained at ~pH 7, suggesting viability for biomedical applications.
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INTRODUCTION
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Wet environments pose challenges for initial contact adhesion due to the presence of a
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superficial hydration layer.1 However, wet conditions do not appear to restrict the marine fouling
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of mussels and barnacles. Mussels and barnacles are major marine biofouling organisms that
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maintain adherence to hard surfaces in environments conventionally hostile to current adhesive
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technologies (Figure 1).2,3 Interfacial mussel foot proteins (mfps), e.g., mfp-3 and mfp-5 (Figure
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2a), are located at the plaque-substratum interface and contain ~20-30 mol% of 3,4-
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dihydroxyphenylalanine (Dopa), a key functional group in the adhesive mechanism of mussel
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plaques.4,5 Mussel-inspired Dopa (or catechol) functionalization has, therefore, been translated to
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synthetic systems with the intent to mimic the strong wet adhesive properties of mussels6,7,8 for
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numerous underwater applications.9,10 However, barnacle adhesive cement proteins, e.g.,
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Megabalanus rosa cement proteins (mrcps), contain non-catecholic aromatic functionalities such
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as tyrosine, tryptophan, and phenylalanine rather than Dopa.11 Mrcp-100k and mrcp-52k (Figure
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2b) appear plentifully in barnacle cement and are presupposed as cooperative agents of adhesive
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cement formation in wet environments.12
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Although the adhesive mechanisms of mussels and barnacles are not yet fully understood,
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instantaneous wet contact adhesion of these proteins is required for the sessile organisms to
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adhere to marine surfaces. Other interfacial interactions contributing to adhesion in these
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organisms, e.g., hydrophobic and electrostatic interactions, are less often translated to synthetic
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systems, as previous studies have often neglected to replicate the structural presence of non-
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Figure 1. Barnacles and mussels attach to a variety of surfaces in intertidal marine environments
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(Campus Point, UC Santa Barbara, Santa Barbara, CA).
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catechol moieties or otherwise integrate only a single electrostatic functionality.13 Nevertheless,
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recent studies incorporating hydrophobic and electrostatic interactions with catechol in
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polyampholytes14 and zwitterionic small molecules15 showed record high underwater adhesion
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energy values up to an order of magnitude greater than previously reported in natural,16
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recombinant,17 or fusion adhesive proteins.18
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Therefore, we sought to study the effects of catecholic and non-catecholic (benzyl) components
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and the contribution of the key functional analogs on initial contact adhesions under various
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conditions, e.g., dry, deionized water, and artificial seawater environments. As such, we
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combined structural motifs from these marine adhesive proteins into a series of bio-inspired UV-
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cured poly(acrylate) films. These synthetic copolymers retain the important functionalities of
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both mfps and mrcps, e.g., aromatic (Dopa, tyrosine, and tryptophan), cationic (lysine, arginine,
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and histidine), anionic (aspartic acid), and non-polar (glycine, leucine, alanine, and proline)
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residues. Initial contact adhesion of the copolymers was measured using a probe tack testing
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apparatus and compared in relation to the proportion of each functional group.
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Figure 2. Color-coded sample sequences of interfacial mfps (a) and mrcps (b), along with the
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key functional groups incorporated in their synthetic analogs.
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EXPERIMENTAL SECTION
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Materials and Methods
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Sample preparation. Benzyl acrylate (BA) purchased from Sigma-Aldrich (USA) and a silyl-
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protected catechol acrylate (CA) provided by Osaka Chemical (Japan) were employed as analogs
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of the benzyl and catechol moieties of natural marine adhesive proteins, respectively.
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Additionally, 2-ethylhexyl acrylate (EHA), 2-(dimethylamino)ethyl methacrylate (DMAEMA),
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and acrylic acid (AA) were purchased from Sigma-Aldrich to serve as analogs of non-polar,
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cationic, and anionic residues, respectively, of the natural proteins. The monomers were used as
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received and mixed in accordance with the molar ratios presented in Table 1 along with 2 wt.%
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of a radical photoinitator (Irgacure 819, Bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide)
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(BASF, USA) dissolved in toluene. The role of the organic solvent was to ensure homogeneous
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distribution of the solid photoinitiator in the formulation. The comonomer mixtures were then
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vortexed to further ensure homogenization. Ratios of AA and DMAEMA were varied in the
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samples at 1:2 and 2:1 ratios. The catechol-containing copolymer films were
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Figure 3. An approach to determine the initial contact adhesion of the UV-cured copolymer
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films under ambient dry and wet testing conditions.
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prepared from a silyl-protected catechol acrylate due to the spontaneous autoxidation of the
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catechol functional group. The silyl-protecting groups were later cleaved in an aqueous pH 1-2
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HCl solution at room temperature to produce a catechol-functionalized acrylate film, based on a
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previously described method.14,19 After immersion for 20 minutes, the samples were removed
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from the acidic solution and dried via air stream.
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Fusion UV System. UV radical polymerization was carried out with the Fusion UV system
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(provided by Heraeus Noblelight America, USA) which consisted of a 300 watt/inch (2.54 cm) H
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lamp and LC6B benchtop conveyor belt. The lamp’s distance from the conveyor belt was 10 cm.
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This UV system recorded UVA Band (320-390 nm) and 615–660 mJ/cm2 of UV radiation dose
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Table 1. Molar percentages of acrylate monomers in the prepared samples. Composition Aromatic Non-polar Ionica Sample 1 0% 50% 50% Sample 2 0% 60% 40% Sample 3 0% 70% 30% Sample 4 0% 80% 20% Sample 5 0% 90% 10% Sample 6 0% 100% 0% Sample 7 10% 50% 40% Sample 8 10% 60% 30% Sample 9 10% 70% 20% Sample 10 10% 10% 80% Sample 11 10% 90% 0% Sample 12 20% 50% 30% Sample 13 20% 60% 20% Sample 14 20% 70% 10% Sample 15 20% 80% 0% b 30% 50% 20% Sample 16 Sample 17 30% 60% 10% Sample 18 30% 70% 0% Sample 19c 40% 50% 10% Sample 20 40% 60% 0% Sample 21d 50% 50% 0% a Ionic ratios of either 1:2 AA:DMAEMA or 2:1 AA:DMAEMA. b-d Samples selected for further analysis.
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at a belt speed of 20 feet/min at focus, as measured by an EIT Power Puck 1 Radiometer
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(Sterling, USA). Neat free radical UV-initiated polymerization was carried out at room
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temperature in air. The dose of UV radiation and the exposure time were controlled by adjusting
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the belt speed and the number of scans.14
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Incubation. Incubation (prepolymerization) of the comonomer mixtures was required to form a
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uniformly thick adhesive film. Each mixture was scanned in vitro at 20 feet/min to produce a
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more viscous solution. Samples in dram vials were fixed to a stainless steel plate and put through
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the UV light system to create a polymer precursor with desired viscosity. The number average
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molecular weight (Mn) of the incubated viscous mixture was ~3000-5000 Da (Table 2), as
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measured by Gel Permeation Chromatography (GPC) (Figure S1).
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Neat UV free radical polymerization. After the incubation process, roughly 300 µL of each
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sample was placed on individual sheets of 50 µm thick Mylar® PET backing (CS Hyde
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Company, USA). A #40 EC-100 wire wound “Mayer” rod (Cheminstruments, USA) was then
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slowly passed over the sample to create a 100 µm thick layer. The PET sheets were then passed
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through the previously described UV system at a belt speed of 10 feet/min, which corresponds to
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a UV dose of 1230-1320 mJ/cm2 per pass. Optimum cohesion and adhesion of the copolymerized
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films was obtained after three passes through the system.
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Dry Probe Tack Test. Once each adhesive film was polymerized and allowed to cool for 5
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minutes, 0.5 cm x 1 cm strips of the copolymer films on PET backing were placed directly on the
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PT-1000 Polyken Probe Tack (PT) Machine (Cheminstruments) stage opening (Figure 3) and
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fixed to the stage with lab tape. Each strip was tested once in ambient dry conditions with a flat-
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ended cylindrical steel probe tip (diameter = 5 mm; contact area ~0.20 cm2) with an approach
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and separation speed of 61 cm/min, contact time (tc) of 5 seconds, and an applied load of 768.9
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mN ± 7.9 mN. All parameters were held constant throughout testing. A representation of the
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typical probe tack test curve is included in the Supporting Information (Figure S2). The area
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underneath the probe tack curves was used to determine the work of detachment (Wad), as
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outlined in a previous study.20
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Wet Probe Tack Test. To investigate the role of the differing aromatic functional groups,
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benzyl group-containing copolymer samples with the highest measured tack strengths (Samples
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16, 19, and 21) (Figure 4) were made again with direct substitution of BA for CA. Both the
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benzyl- and catechol-containing copolymers were then tested under wet conditions (Figure 5).
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75 µL of deionized (DI) water or artificial seawater was placed on the probe tip surface prior to
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sample contact with the probe tip, and probe tack strength was then measured. Additional PT
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testing was then completed under a range of various pH conditions (Figure 7), utilizing an
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identical set up to the previous wet PT tests. Solutions of pH 3, 7, 9, and 11 were used to test the
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adhesives. DI water was used for the pH 7 environment, acetic acid was used to decrease pH of
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DI water to 3, and sodium carbonate was used to increase pH of DI water to 9 and 11.
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Probe Tack Test for Commercial Tapes. In order to establish the relative probe tack strength
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of our selected samples, additional PT tests were performed with two commercially available
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PSAs, Fisherbrand™ Colored Label Tape (Fisher Sci, USA) and Scotch® Packaging Tape (3M,
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USA) under both dry and wet conditions. These adhesives were adhered directly onto the PT
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stage, with stage placement, tc, approach speed, applied load, and separation speed kept
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consistent with the aforementioned parameters.
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Statistical analysis. The Mann-Whitney U test was used to compare the median probe tack
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strengths of different treatments. A statistically significant difference was determined when p-
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values were less than 0.05.
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RESULTS AND DISCUSSION
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A total of forty-one copolymer samples were prepared with differing ratios of anionic, cationic,
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non-polar, and aromatic functional groups (Table 1). Samples containing greater proportions of
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aromatic residues exhibited increasing probe tack strength. Samples 16, 19, and 21, containing
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30-50% aromatic, 0-20% charged, and 50% non-polar groups, showed greater dry probe tack
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strength, regardless of charged acrylate composition (Figure 4), and were selected for further
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studies.
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Despite the absence of crosslinking agents in the formulations, the polymers exhibited sharp
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increases in viscosity and were insoluble in organic solvents after polymerization. The reduced
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mobility of free radicals along the polymer backbone resulted in more proximal free radical side
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branching as well as a decrease in the termination rate. As a result, these side branches generated
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interchain crosslinkages, leading to the formation of a polymer network in a process known as
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the Trommsdorff-Norrish effect.21,22
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Due to this effect, GPC results (Table 2) were obtained from linear, non-crosslinked portions of
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the polymers, as insoluble, crosslinked components must be filtrated prior to analysis. For this
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reason, GPC results obtained from samples after incubation did not differ from the results
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obtained after polymerization. The presence of this gel effect also prevented other forms of
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analysis, e.g., H-NMR, after polymerization.
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Neat UV polymerization generates broad molecular weight distributions that enable deformation
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of acrylate polymers on rough surfaces so as to improve tack properties.23 Acrylic PSAs are
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produced without purification or washing as to maintain this broad range. PSAs require not only
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broad molecular weight dispersity21 but also lower Tg values in order to adequately wet the
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substrate and create tackiness at bonding sites. Our samples exhibited high polydispersity and Tg
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ranging from -27.5 °C to -9.1 °C (Table 2 and Figures S10-12).
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Table 2. Molecular weights, polydispersity, and glass transition temperatures of selected
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incubated mixtures.
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Sample 16 Sample 19 Sample 21
Mn (g/mol) 2730 5064 3749
Mw(g/mol) 12787 66424 82279
Đm (Mw/Mn) 4.7 13.1 22.0
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Tg(°°C) -19.4 -27.5 -9.1
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Attenuated Total Reflection Infrared Spectroscopy (ATR-IR) was used to monitor UV radical
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polymerization as a function of increasing UV dose in proportion to the number of UV scans.
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ATR-IR analysis (Figures S3-S5) showed a decrease in the intensity of peaks attributed to C=C
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acrylate double bonds, found at 810 cm-1 (Figure S6) and 1410 cm-1, after prolonged exposure to
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UV radiation, suggesting successful UV-initiated radical polymerization. Gas Chromatography
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(GC) was also used to monitor monomer consumption and polymerization. GC (Figures S6-S8)
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revealed that there
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Figure 4. An initial comparison of dry probe tack strengths from samples with differing ionic
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charge ratios. Error bars indicate standard deviation, n=5.
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were only trace amounts of unreacted monomers, and no residual solvent after UV
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polymerization, suggesting polymer compositions nearly equal to the feeding monomer ratio.
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Copolymer samples with a 2:1 ratio of anionic to cationic groups afforded greater probe tack
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strength than cationic-dominant alternatives in ambient dry conditions (Figure 5). Samples
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prepared with a 2:1 AA:DMAEMA ratio displayed probe tack strengths of 19.4 ± 5.5 N/cm2
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(Sample 16) and 23.8 ± 3.1 N/cm2 (Sample 19), greater than the results obtained from
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formulations prepared with a 1:2 AA:DMAEMA ratio, 15.2 ± 1.7 N/cm2 (Sample 16) and 22.3 ±
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2.0 N/cm2 (Sample 19) (Figure 5). The work of detachment values, Wad, were 18.3 ± 2.2 mJ/cm2
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for Sample 16 and 24.3 ± 2.9 mJ/cm2 for Sample 19 prepared with 2:1 AA:DMAEMA, whereas
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the values obtained from samples prepared with 1:2 AA:DMAEMA were 13.3 ± 1.8 mJ/cm2 for
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Sample 16 and 23.8 ± 3.7 mJ/cm2 for Sample 19 . These results are consistent with the
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composition of most commercial PSAs made for dry adhesion, which contain ~10% anionic
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residues. Conversely, mfp-5, the most wet-adhesive natural protein, contains a 1:2 ratio of
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anionic to cationic residues. Copolymer samples with 1:2 ratio of anionic to cationic residues
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showed greater wet probe tack strengths than anionic-dominant films in artificial seawater
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environments. Interestingly, Sample 21, lacking ionic functionalities, exhibited even greater wet
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probe tack adhesion values in DI water (15.2 ± 1.4 N/cm2) and seawater (8.7 ± 1.0 N/cm2).
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Sample 21 also exhibited comparatively large work of detachment values in DI water (Wad =
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15.3 ± 1.9 mJ/cm2) and seawater (Wad = 7.5 ± 0.3 mJ/cm2). Samples 16 and 19 with cationic-
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dominant compositions and Sample 21 were consequently selected for further analysis due to
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their appreciable probe tack strengths in artificial seawater.
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Figure 5. A comparison of probe tack strengths of selected copolymer films under ambient dry
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and wet conditions. Error bars indicate standard deviation, n=5.
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In order to investigate the effects of catechol moieties on initial contact adhesion, the samples
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were then prepared with catechol acrylate (CA) in place of benzyl acrylate (BA). The probe tack
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test results from the catechol-functionalized copolymers were then compared to benzyl-
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functionalized copolymers (Figure 6). The median probe tack strengths of the BA and CA
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samples in DI water were 13.6 N/cm2 and 10.5 N/cm2, respectively; the distribution of the two
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groups was not found to differ significantly (Mann–Whitney U = 42, n1 =12; n2 = 11, P > 0.05,
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two-tailed). The median probe tack strengths of the BA and CA samples in artificial seawater
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were 5.5 N/cm2 and 4.3 N/cm2, respectively; the distribution of the two groups did not differ
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significantly (Mann–Whitney U = 91, n1 = 14; n2 = 9, P > 0.05, two-tailed). With the exception
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of the dry probe tack strength of Sample 21 (34.3 ± 3.2 N/cm2) and its work of detachment (Wad
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= 29.5 ± 3.7 mJ/cm2), the probe tack strengths of the benzyl-functionalized samples were
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comparable to the catechol-functionalized samples across all other testing conditions. These
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results suggest that the benzyl-functionalization, though comparatively inexpensive, might offer
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similar wet adhesion performance to catechol alternatives.
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Figure 6. A comparison of probe tack strengths of benzyl-functionalized copolymers, catechol-
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functionalized copolymers, and commercial PSAs under ambient dry and wet conditions. Error
3
bars indicate standard deviation, n=3-15.
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Fisherbrand™ Colored Label Tape and 3M™ Scotch Packaging Tape were selected as
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commercial PSA controls for wet contact adhesion because they exhibited a comparable range of
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dry probe tack strengths (14.2 – 21.1 N/cm2) to our bio-inspired benzyl films (12.3 – 24.3 N/cm2)
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(Figure 6). However, when subjected to ambient wet conditions, commercial adhesives were
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found to have tack strength values (0.1 – 3.2 N/cm2) and work of detachment values (Wad = 0.2 –
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2.6 mJ/cm2), up to 150 times smaller than our benzyl-functionalized Sample 21 (15.2 N/cm2, Wad
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= 15.0 mJ/cm2). The median probe tack strengths of the BA samples in DI water was 13.6
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N/cm2, while the median values for both Scotch Packing Tape and Fisher Lab Tape was ~0.5
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N/cm2. The difference in probe tack strengths was determined to be statistically significant
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(Mann–Whitney U = 0, n1 = 15; n2 = 11, P < 0.05, one-tailed). Additionally, while the benzene
14
functionalized samples were seen to have probe tack strengths approaching 9.0 N/cm2 in ambient
15
seawater conditions, the commercial alternatives produced near-zero probe tack values.
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The samples were also tested across a range of different pH conditions (Figure 7). While the
17
samples presented substantial wet probe tack strengths at neutral pH (12.9 ± 0.94, 8.3 ± 1.0
18
N/cm2 and 15.2 ± 1.4 N/cm2 from Samples 16, 19, and 21, respectively), the initial contact
19
adhesion values were negligible at pH 3 and pH 11. Benzene functionalization might therefore
20
be suitable for biomedical applications near the physiologically relevant pH ~7.
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Figure 7. A comparison of probe tack values of benzyl acrylate copolymer samples in varying
3
pH conditions. Error bars indicate standard deviation, n=5.
4 5
CONCLUSION
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In this study, we prepared copolymer films functionalized with aromatic, non-polar, and/or ionic
7
moieties as analogs of the residues found in mfps and mrcps, and investigated their contact
8
adhesion strengths in relation to the proportion of each residue. The enhanced probe tack
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strength of the marine bio-inspired adhesive films over current commercial pressure-sensitive
10
adhesives demonstrates the viability of aromatic functionalization for conveniently engineered
11
adhesive films with greater wet contact adhesion. Furthermore, benzyl-functionalized copolymer
12
films exhibited similar performance to catechol-functionalized copolymer films without concerns
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about the inherent oxidative instability of catechol residue.
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AUTHOR INFORMATION
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Corresponding Authors
17
*Email:
[email protected]
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Author Contributions
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BKA conceived and designed the research. BKA and SKC wrote the manuscript. §These authors
3
equally contributed to this work.
4
Notes
5
The authors declare no competing financial interests
6 7
Supporting information
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Gel Permeation Chromatography; Probe Tack Test curve; Gas Chromatography; Attenuated
9
Total Reflection Infrared spectra; Differential Scanning Calorimetry. This material is available at
10
free of charge via the Internet at http://pubs.acs.org.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge support from the Office of Naval Research N000141310867
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and the Korea Institute of Industrial Technology. The authors also wish to thank Claus D.
14
Eisenbach (Institute for Polymer Chemistry - University of Stuttgart) for his comments during
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the revision of this manuscript.
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ABBREVIATIONS
17
AA, acrylic acid; ATR-IR, Attenuated Total Reflection Infrared Spectroscopy; BA, benzyl
18
acrylate; CA, catechol acrylate, DI, deionized water; Đm, mass dispersity; DMAEMA, 2-
19
(dimethylamino)ethyl methacrylate; Dopa, 3,4-dihydroxyphenylalanine; DSC, Differential
20
Scanning Calorimetry; EHA, 2-ethylhexyl acrylate; GC, Gas Chromatography; GPC, Gel
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Permeation Chromatography; mfp, mussel foot protein; Mn, number average molecular weight;
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mrcp, Megabalanus rosa cement protein; Mw, mass average molecular weight ; PET, polyester
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film; PSA, pressure-sensitive adhesive; PT, Probe Tack; tc, contact time; Tg, glass transition
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temperature; UV, ultraviolet; Wad, work of detachment.
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