High-performance mussel-inspired adhesives of reduced complexity

Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m−2) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule.

M arine mussels attach to rocks by way of thread-like tethers on wind-and wave-swept seashores where wave velocities can reach 25 m s À 1 . Mussel attachment tenacity is enabled by adaptations at multiple length scales 1 , and the nanometre scale corresponds most closely to the chemistry of the interface 2 . Interfacial mussel foot proteins (mfps), especially mfp-3 and mfp-5, are rich in the catecholic amino acid, 3,4-dihydroxyphenylalanine (Dopa), which has been adopted for functionalizing diverse synthetic polymers 3,4 . Although the role of Dopa in wet adhesion is under increasing scrutiny, its introduction into polymers often endows them with strong underwater adhesion 2,4,5 and self-healing [6][7][8] capabilities.
Other common amino acids, for example, lysine, phosphoserine and histidine, are also relevant to mfp adhesion 9,10 and merit translation to synthetic systems 4,11 . Mfps are protein polyelectrolytes with high charge densities 12 , and lend themselves to complex coacervation, which appears to be critical for the lossless fluidic delivery of mfps to target surfaces underwater 9,12 . Lysine and phosphoserine have been adopted to enhance adsorption 11 , but also to drive charge-dependent complexation 13 needed for the dense fluid phase separation (coacervation) of adhesive polyelectrolytes from the equilibrium solution. Of particular interest here, mfp-5 and -3 function as adhesive primers 14 at the plaque-substratum interface 15 . Despite recent progress in the science of wet adhesion, practical underwater adhesion remains limited or non-existent for a variety of potential applications. Biomimetic synthetic initiatives have aspired to remedy this by mimicking specific attributes of the bioadhesive model systems of mussels and sandcastle worms. These attributes include macromolecular adsorption to surfaces 2,3,16,17 , fluid-fluid phase separation (coacervation) 13,18 and curing by oxidative catechol-based crosslinking 2,19,20 , and involve many complex components and steps requiring optimization and stabilization 21 .
In this study, both the strong wet adhesion 22 of mfp-5 films and the coacervation 9 of mfp-3 were translated into a smaller and simpler zwitterionic platform. We sought to simplify and combine several motifs borrowed from the interfacial adhesive mfps (including catechol, positive and negative charges, and nonpolar moieties) into a single, low-molecular-weight, onecomponent adhesive system. By taking a previously reported gemini zwitterionic surfactant 23 with charged quaternary amine and phosphate groups and two alkyl tails, we replaced one of the tails with a catechol, and varied the other tail to create a family of mfp-mimetic zwitterionic adhesive homologues (mass o500 Da). These homologues exhibit very strong and spontaneous catecholmediated binding to surfaces, stable phase separation as onecomponent coacervates or bilayer films, and periodate-mediated curing. The measured adhesion energy (W ad B50 mJ m À 2 ) is the highest reported to date 22,24 for a nm-thick film formed underwater, and 2-3 times greater than mfp-5 (ref. 22) or the recently engineered recombinant mfp-amyloid fusion protein 24 . Zwitterion-mediated adhesion is likely to stimulate applications at multiple length scales including nanofabrications 25,26 that require molecularly smooth and thin (o4 nm) adhesive layers, for example, in electronic 27 , lithographic 28 and biomedical applications 2 .

Results
Zwitterionic adhesives design inspiration. The phosphate, amine, catechol and hydrophobic (alkyl or benzyl) functionalities in the zwitterions were adopted from the following mole percents of amino-acid residues in mfp-5, for example, anionic residues   (B15 mol%), cationic residues (B29 mol%), Dopa (B27 mol%) and assorted non-charged (B29 mol%) ( Fig. 1) 22 . Accordingly, the chemical complexity of mfps was reduced, and adhesion and coacervation were recapitulated by a strategic combination of functionalities in the zwitterionic platform (Fig. 1e,f). Various homologues were designed by replacing one or both alkyl chains with mfp-mimetic functionalities (Fig. 2a) and their interfacial and adhesive properties were investigated.
Interfacial properties and redox stability. The aggregation tendency of each homologue in water was assessed by the critical aggregation concentration (CAC). The CAC is the concentration at which further addition of solute molecules to a solvent makes them go into finite-sized aggregates while the monomer concentration remains unchanged 29 . The CAC was determined from the solution surface tension of varying homologue concentrations in deionized water (NB the red line of Z-Cat-C10 in Fig. 2b) by Wilhelmy plate tensiometry. Figure 2b shows that decreasing alkyl tail length increases the CAC gradually from Z-Cat-C10 to -C8, -C6 and -C4 (parabolic dashed line drawn in light blue from Z-Cat-C10 to -C4), consistent with thermodynamic predictions 29 . Conversely, aromatic substitution ( Fig. 2a) increased CAC significantly. The substitution of the catechol of Z-Cat-C8 by a benzene (Fig. 2a) also increased CAC. On the basis of cyclic voltammetry combined with ultraviolet-visible spectroscopy, the oxidative stability of catechol functionalities in all zwitterionic homologues (for example, E ½ of Z-Cat-C10B þ 0.25 V at 1 mV s À 1) was significantly increased by comparison with 4-methylcatechol (completely solvent accessible, DE ½ B þ 0.12 V in deionized water at 1 mV s À 1), suggesting a highly stabilizing environment in the dispersion ( Supplementary Fig. 1).
Surface force measurements and interfacial interaction study.  2e). Notably, the interaction energies of Z-Cat-C10, -C8, -C6 and -C4 were closely correlated with the CAC (light blue parabola drawn in the plot in Fig. 2e as in Fig. 2b). The hard-wall thickness (the limiting distance between the mica surfaces during the approach run in the SFA) of each homologue was measured and correlated with the CAC (Fig. 2f) corresponding to the catechol-containing zwitterions, which formed strongly cohesive (W c ¼ 2.2-21.6 mJ m À 2 ) bilayers (0.5-2.0 nm). In contrast, the non-catecholic Z-Ben-C8 formed multilayered films (B5 nm) without cohesive tendencies. In the catechol-containing homologues with alkyl tails (Fig. 2a), for example, Z-Cat-C10, -C8, -C6 and -C4, the hard-wall thickness and CAC were both correlated with alkyl tail length (light blue line in Fig. 2f).
Z-Cat-C10, which exhibited the lowest CAC among the homologues and readily phase separated into dense visible coacervates and equilibrium solution at high concentrations (100 mg ml À 1 ), was chosen for further scrutiny (Fig. 3a). Coalescence of the coacervate microdroplets in bulk solution was observed by microscopy ( Fig. 3b and Supplementary Movie 1), and spreading of coacervates over a glass substrate was recorded on an inverted microscope (Supplementary Movie 2). Cryogenic transmission electron microscopy (cryo-TEM) images ( Fig. 3c and Supplementary Figs 13 and 14) showed small micelle-like aggregates in both equilibrium solution and coacervate phases.
Mussels rely on quinone-mediated protein crosslinking to increase plaque cohesion. o-Quinones are produced by the twoelectron oxidation of targeted Dopa residues 22 . Accordingly, we oxidized the catechol in Z-Cat-C10 with sodium periodate resulting in a stoichiometric À 2H þ and À 2e À oxidation to o-quinone 19 . Given the nanoNewton precision of the SFA, we measured the adhesion of monomolecular layers of Z-Cat-C10 on mica. SFA force-distance profiles between surfaces allow the measurement of adhesion forces that can be translated into adhesion energies (W ad ) by the Johnson-Kendall-Roberts theory for soft deformable surfaces 29,30 . Analysis by SFA showed that the observed cohesion or adhesion energy correlated well with the symmetric or asymmetric oxidation of catechol by periodate 7 . After symmetric periodate oxidation (Fig. 4a), cohesion initially decreased by 50% for contact times of rt c ¼ 10 min, but then rebounded after t c ¼ 60-min contact times. Conversely, asymmetric oxidation, that is, all catechol on one side and all quinone on the other (Fig. 4b), maintained the initial adhesion for contact times of rt c ¼ 10 min, followed by a 50% increase in adhesion after a t c ¼ 60-min contact time. The loss 22,24 and subsequent recovery 22 of adhesion, respectively, following oxidation were previously explained as the loss of H-bonding interactions because quinones are exclusively H-bond acceptors (short term), followed by the quinone-mediated formation of covalent crosslinks (long term). The high adhesion (W ad B50 mJ m À 2 ) obtained after t c ¼ 12 h of compressive contact, contrasts with rt c ¼ 10 min and t c ¼ 1 h contacts. According to the post-oxidation analyses, covalent crosslinks formed within and between the thin films (B2-4 nm) (Fig. 4c).
Not limited to mica (Fig. 4d), 5 mM Z-Cat-C10 formed thin, molecularly smooth (B2 nm) films on silicon wafers ( Supplementary Fig. 15) and copper ( Supplementary Fig. 16). The wet and dry adhesion of these films (Supplementary Figs 17-20) on silicon wafers (SiO 2 , root mean squared (r.m.s.) surface roughness o1 nm) and copper (CuO 2 /Cu(OH) 2 , r.m.s. surface roughness B2 nm) surfaces was measured using silica (SiO 2 ) probes in the AFM (Fig. 4e). The surfaces were prepared by spreading 5 mM Z-Cat-C10 over the surfaces, followed by thorough rinsing and adhesive testing (Fig. 4f). The wet work of adhesion (W ad ¼ F ad /1.5pR), B7.7 mJ m À 2 for the SiO 2 and SiO 2 interface, resembled W ad B7.0 mJ m À 2 reported for catecholfunctionalized polysiloxanes and SiO 2 interface 31 , and B0.1 mJ m À 2 for adhesion between the CuO 2 /Cu(OH) 2 and SiO 2 surfaces. After air drying the surfaces for B5 min, the adhesion (W ad ) between CuO 2 /Cu(OH) 2     ARTICLE increased significantly to B4 mJ m À 2 and to B175 mJ m À 2 between SiO 2 and SiO 2 surfaces, which, in the latter case, is 50-fold greater than the W ad (B3.5 mJ m À 2 ) for catecholic polysiloxane adhesion to SiO 2 as calculated from the F ad (83 nN) measured using silica probes (f ¼ 5 mm) in AFM 31 . Comparatively weaker adhesion to the copper plate is tentatively attributed to higher r.m.s. and hydroxides on its surface measured by AFM and X-ray photoelectron spectroscopy (XPS), respectively (Supplementary . We also prepared a suspension of silica beads (f ¼ 100 nm) in deionized water, and spread it over a silicon wafer previously coated with 5 mM Z-Cat-C10. The silica beads remained glued to the film, even after rinsing with pulsed jets of running tap water (0.24 MPa; Fig. 4g).

Discussion
Catechol-functionalized zwitterionic surfactants are clearly more than detergents, and show the following favourable attributes as adhesive primers: (1) they aggregate in water as dense fluidic coacervates at concentrations above the CAC; (2) the coacervates spread uniformly over surfaces; (3) in confinement in the SFA, the coacervates rearrange as paired bilayers (B2.5-nm thickness) with the catecholic head groups facing the mica surface on one side and bulk solvent on the other; (4) the coacervate environment stabilizes catechols by B þ 0.1 V against oxidation; and (5) following periodate treatment, adhesion of thin films of Z-Cat-C10 on mica approaches W ad B50 mJ m À 2 -a new high value for catechol-mediated adhesion.
The results reveal Z-Cat-C10 adhesion on mica to be two-to three-fold greater than mfp-5 (14 mJ m À 2 ) (ref. 22) and the new mfp-amyloid recombinant fusion protein (21 mJ m À 2 ) (ref. 24). Results obtained before and after periodate oxidation of Z-Cat-C10 suggest the following failure modes: before periodate, the work of adhesion averaged B20 mJ m À 2 . Because the hydrophobic bilayer and catechol-to-mica contacts are strong, the weaker inter-catechol H-bonding between the two bilayers ( Fig. 4c) unzips during separation. After covalent arylaryl coupling with periodate, the adhesion failed at B50 mJ m À 2 , which is commensurate with the energy required to separate the two halves of a lipid bilayer leaflet 32 , thus suggesting that adhesion to the mica surface is stronger than B50 mJ m À 2 .
Because the SFA-based work of adhesion (units J m À 2 ) is difficult to relate to reported bulk adhesion properties (J m À 3 ), we shall compare it with peel fracture energy. With native mussel plaques deposited onto silica, peel fracture energy 1 was 100 J m À 2 , which resembles the energy of high-performance adhesive polystyrene; polyvinylpyrrolidone (PS/PVP) blends on silicon 28 . The magnitude of disparity between the work of adhesion and peel fracture energy is striking but plausible. In tests of symmetrically deposited Z-Cat-C10, the paired bilayer structure (Fig. 4c) offers little opportunity for energy dissipation via sacrificial bonds and hidden lengths 33 , whereas in the plaque, molecular scaffolds of different length scales exist 34 . The relationships between the adhesive surface primers and their overlying molecular scaffolds are not well understood. Although the catechol-functionalized zwitterionic surfactants were designed to be adhesive primers, some bulk tests in lap shear or a threepoint bending peel mode on stainless steel (SS304) (Supplementary Fig. 12) showed unexpected gap-filling capabilities and strengths of B1 MPa or B20 N cm À 1 , suggesting that stacked bilayers as shown in Fig. 4c may not be limited to two layers.
In summary, we show that a synthetic low-molecular-weight catecholic zwitterionic surfactant mimics the strong adhesive priming and self-coacervating properties of mfps. The high wet-cohesion (or -adhesion) energies ranged from B20 to B50 mJ m À 2 for uncrosslinked and crosslinked films, respectively. This study underscores the importance of catechols and of maintaining a balance 4 between hydrophobic and electrostatic interactions for tuning or optimizing both coacervation and adhesion. These catecholic zwitterions are adaptable as coatings or adhesive primers 14,28 for diverse surfaces and, given their uniformly thin (o4 nm) and strong glue layers, hold particular promise as a new tool for nanofabrication [25][26][27]35 (please see Supplementary Note and Discussion for appropriate details).

Methods
Synthesis. The zwitterionic surfactants were synthesized based on a previously described procedure 23

(Supplementary Methods).
Critical aggregation concentration. Aqueous colloidal dispersions of each homologue were prepared in deoxygenated deionized water. The CAC was determined from the solution surface tension of varying homologue concentrations in deionized water (for example, see the red line of Z-Cat-C10 in Fig. 2b) by Wilhelmy plate tensiometry and has been described elsewhere 36 . Use of the CAC instead of Critical Micelle Concentration (CMC) here is a hedge to accommodate our uncertainty about whether micelles or other soluble aggregates were forming.
Cyclic voltammetry. Cyclic voltammetry (CV) was performed on a Versastat 3 potentiostat from Ametek Co. (Berwyn, PA). These analyses were carried out using a three-electrode cell: a Pt wire as the counter electrode; an Ag/AgCl reference electrode; and a carbon paste electrode (CPE) served as the working electrode. Carbon paste electrode is the best choice for adhesive materials because of its ability to be completely polished before each experiment.
Measuring adhesion and cohesion energies. The adhesion/cohesion energy of the synthetic zwitterionic molecules was investigated in SFA (SurForce LLC, Santa Barbara, CA). Two molecularly smooth freshly cleaved back-silvered mica surfaces glued on cylindrical silica discs of radius of curvature, RB2 cm were mounted in the SFA and 50 ml of a colloidal dispersion of the synthetic molecules in deionized water was injected between the surfaces at different concentrations (C ¼ 0.5-5 mM) and the forces between the thin films of the molecules were measured by multiple beam interferometry 37 . The adhesion forces between Z-Cat-C10 molecules on various surfaces, for example, silica and copper, were also measured by AFM (MFP-3D, Asylum Research, Goleta, CA) in deionized water and ambient conditions, respectively, at room temperature. AFM probes with a silicon dioxide particle (10, 5 and 1 mm, respectively) on a silicon nitride (SN) cantilever À 0.6 N m À 1 were purchased from Novascan Technolgies, Inc. (Ames, IA). Cantilever spring constants were measured for each force run. Images of Z-Cat-C10 coating on silicon wafer and copper plates were visualized in an AFM.
Cryo-TEM. Cryo-TEM vitrified specimens were prepared in a controlled environment vitrification system at 25°C and 100% relative humidity. The procedure is described in detail elsewhere 38 . About 3 ml of the sample was applied onto a perforated (lacey) carbon film-coated 200 mesh copper TEM grid. Excess liquid was blotted with filter paper, and the specimen was immediately plunged into liquid ethane at its freezing point ( À 183°C). The vitrified specimens were 240 transferred to a 626 Gatan cryo-holder, using it tansfer station, and imaged at 120 kV acceleration voltage in an FEI Tecnai T12 G 2 TEM, using low-dose imaging to minimize electron-beam radiation damage. Typical electron exposures were 10 to 15 e À Å À 2 . The specimens were kept in the TEM at about À 175°C. Images were digitally recorded with a Gatan US1000 high-resolution (2it Â 2highresolution (2charge-coupled device camera, using the Digital Micrograph software. XPS. XPS (Kratos Axis Ultra, Kratos Analytical, Manchester UK) was conducted with survey scans at a pass energy of 160 eV, and high-resolution scans at a pass energy of 40 eV. Nano-layer adhesion to silica surface was imaged with SEM after rinsing silica bead (diameter j ¼ 100 nm) dispersion (10 mg ml À 1 in deionized water), spread on a silicon wafer coated with Z-Cat-C10, thoroughly with deionized water.