Bioinspired catechol-terminated self-assembled monolayers with enhanced adhesion properties

The role of the catechol moiety in the adhesive properties of mussel proteins and related synthetic materials has been extensively studied in the last years but still remains elusive. Here, a simplified model approach is presented based on a self-assembled monolayer (SAM) of upward-facing catechols thiol-bound to epitaxial gold substrates. The orientation of the catechol moieties is confirmed by spectroscopy, which also showed lack of significant amounts of interfering o-quinones. Local force-distance curves on the SAM measured by atomic force microscopy (AFM) shows an average adhesion force of 45 nN, stronger than that of a reference polydopamine coating, along with higher reproducibility and less statistical dispersion. This is attributed to the superior chemical and topographical homogeneity of the SAM coating. Catechol-terminated SAMs are also obtained on high-roughness gold substrates that show the ability to assemble magnetic nanoparticles, despite their lack of enhanced adhesion at the molecular level. Finally, the influence of the catechol group on the formation and quality of the SAM is explored both theoretically (molecular dynamics simulations) and experimentally using direct-write AFM lithography.


Introduction
Polydopamine (PDA) has emerged in recent years as a highly versatile bio-inspired coating and adhesive primer, after the pioneering research by Messersmith and co-workers. [1] These authors described an easy and straightforward coating method based on the aerobic autooxidation of dopamine in mildly basic aqueous media, and simultaneous in situ deposition of the resulting polymer. [2] Such methodology was shown to be effective on a wide variety of substrates, ranging from inorganic (e.g. metals, metal oxides) to organic (e.g. polymers), including notoriously difficult materials, such as PTFE, [3] with a remarkably wide-ranging effectiveness. [4] Since this seminal work, the possibilities of polydopamine as primer coating have been thoroughly explored and used in cell adhesion, [5] nanoparticle coating and assembly, [6,7] drug delivery, [8] and membrane modification [9] among others. Moreover, alternative synthetic approaches to obtain polydopamine-like materials with similar coating efficiencies have also been reported. [10] Such adhesion properties are mostly attributed to the catechol moieties present in the PDA structure. Notwithstanding its usefulness, the structure of PDA is still subject of debate, being consistent with an association of chemically disordered oligo-/polymeric scaffolds containing both catecholic and o-quinoid moieties. [11] Its structural disorder, together with the typical irregular topography of polymeric coatings, makes it very difficult to gain details on the PDA surface characteristics, which are generally considered to be determined by the outermost 5-10 Å layer. [12] In this context, catechol-based self-assembled monolayers (SAMs) arise as a promising research alternative to establish meaningful structure-property relationships.
SAMs are molecular assemblies of organic adsorbates that spontaneously organize on surfaces in an orderly fashion by an easy adsorption process. [13] Head groups self-assemble together on suitable surfaces forming domains of close-packed molecules where the tail (outward facing) groups determine the characteristics of the coated surface. Previous examples of catechol-terminated SAMs have been reported so far, [14] though most of the work focuses on their electrochemical behaviour. [15] As far as we know, the adhesive properties of catechol-terminated monolayers are yet to be quantified and studied in detail. Herein we report the study of SAMs of 4-(6'-mercaptohexyl)catechol (1)  that the catechol group has on the structure and formation of the monolayer.

Synthesis and self-assembly of Compound 1
The synthesis of target thiol 1 was achieved through a convergent synthetic approach, as shown in Scheme 2. The aldehyde moiety of commercially available 3,4dibenzyloxybenzaldehyde was reduced with NaBH 4 to give the corresponding alcohol, which was then treated with PBr 3 to afford the bromoderivative 2 in 89% overall yield. Afterwards, compound 2 was quantitatively transformed in the corresponding phosphonium bromide salt following a reported procedure. [16] On the other hand, the already described 5-bromopentanal was obtained from 5-bromobutanol in 87% yield by oxidation with PCC. [17] Combination of the two fragments in a Wittig reaction, using K 2 CO 3 as a base, gave a 1:1 mixture of olefins  Figure S3). The resulting catechol-terminated SAMs were characterized using Polarization Modulation-Infrared Reflection-Absorption Spectroscopy (PM-IRRAS), X-Ray Photoelectron Spectroscopy (XPS) and Spectroscopic Ellipsometry. The PM-IRRAS spectrum (see Supporting Information, Figure S1 and Table S1) is in agreement with those previously reported for related catechol-terminated SAMs. [18, 14c-d, 13a] Vibrational bands ranging from 2960 to 2855 cm -1 , which are also present in the corresponding ATR spectrum of a bulk sample, are assigned to the stretching modes of C-H bonds in the alkyl chain. Importantly, the band around 2495 cm -1 assigned to the S-H bond stretching is not observed in the monolayer spectra, fully consistent with thiol groups covalently bound to the Au substrate. The presence of the hydroxyl groups is confirmed by the peaks at 3462 cm -1 , 1264 and 1113 cm -1 , assigned to O-H stretching and in-plane bending, and C-O stretching modes. Peaks at 1522 and 1458 cm -1 are assigned to the in-plane stretching of the C=C bonds of the aromatic ring. [19] Overall, the high intensity of the signals associated to the C-O, O-H, and C=C bonds discard the possibility of a parallel orientation of the aromatic ring with regard to the surface -which would be expected if the adsorbates were lying flat on the substrate-, and hence suggest that the thiols are standing in an orderly fashion with a certain tilt angle. [20] Finally, a very weak signal appears at 1666 cm -1 , indicating the presence of traces of oxidized (o-quinoid) catechol species.
XPS results are also in agreement with those found for similar catechol-terminated monolayers [14d, 17] (See Supporting Information, Figure S2). The S 2p core level clearly presents two peaks with a 2:1 ratio, located at 161.9 and 163.0 eV, assigned to sulphur atoms chemically bound to gold surfaces. A more detailed deconvolution of this spectral region suggests that a small amount (less than 10%) of unbound sulphur species may also be present in the sample. Most likely, these signals arise from disulfides that spontaneously generate when the thiol is in solution, as observed during the synthesis of 1. The XPS C 1s core level is deconvoluted into three peaks at ~284.3, 284.5 and 286.0 eV (peak ratio 2:3:1), corresponding to four aromatic H-bound carbons, six aliphatic, and two O-bound aromatic carbons, respectively. Although oxidation of the catechol moiety to o-quinone is feasible in an oxygen atmosphere, no significant contribution from this moiety is observed by XPS, in accordance with PM-IRRAS spectra. Importantly, the ratio of intensities between the C 1s and the S 2p signals measured on the same sample at different take off-angles (TOA) increases when the measurement is performed at TOA=60º . This indicates that S atoms are preferentially located close to the surface, while C atoms tend to position away from it, in agreement with what should be expected for a S-bound monolayer. [21] Finally, the estimation of film thickness was performed using spectroscopic Ellipsometry in the range between 300 -400 nm. According to its UV-Vis spectra, 1 does not absorb light in this wavelength range, so we chose to model the film with a transparent medium with a refractive index of 1.49. [22,23 ] Film thickness was found to be approximately 5 Å, being slightly dependent on the point of sampling. Since this value is well below the full-stretched length of the molecule -ca. 12 Å-, the formation of stacked multilayers was ruled out. Using this same molecular length value, an average tilt angle with respect to the surface of ca. 27 degrees may be calculated.

Adhesive Properties and Surface Effects
The adhesive properties of the catechol-terminated SAMs on epitaxial gold were studied by AFM force-distance (F-d) curves, a technique that had already been used to study the adhesive properties of dopamine. [24] In a typical experiment, a non-functionalized AFM tip is brought into contact with a surface at a constant speed and then pressed against it to a fixed load; afterwards, the tip is retracted from the surface. During the whole process, the deflection of the cantilever is registered and plotted as a function of the extension of the piezoelectric sensor. Depending on the information to be derived from the experiments, different parts of the F-d curves should be analyzed. [25] In our case, adhesion force values between the tip and the sample were calculated from the jump-out of the tip during the retraction movement (see Supporting Information, Fig. S3 and the Experimental Section for more details).
Representative histograms of adhesion force were constructed from repeated force-distance curves registered across the surface. Two additional substrates -bare epitaxial gold and gold modified with an ODT monolayer-, were also studied for comparison purposes. The experiments were performed on the same experimental session, in order to minimize temperature and humidity fluctuations.
Bare gold substrates presented a force histogram centred at small values of 8-9 nN (see Overall, the catechol-terminated monolayer showed an average adhesion force at the nanoscale level five times higher than that measured for bare gold, and eight times higher than that of an ODT monolayer. -Insert Figure 1 here-For comparison purposes, additional F-d measurements were recorded on a polydopamine coating obtained following the procedure already described in the literature. [1] Epitaxial gold substrates were kept in vertical orientation while immersed in a stirred aqueous solution of dopamine hydrochloride (pH 8.5) for an hour and then rinsed with Milli-Q water (see Experimental Section for more details). AFM imaging of PDA-coated substrates revealed a rough topography formed by small aggregates deposited on the surface, in agreement with previously reported data [27] (see Supporting Information Figure S4). Afterwards, F-d curves were recorded and represented in the histogram shown in Figure 2. In some experiments, PDA coatings showed multiple jump-off or deformed curves with a high degree of statistical dispersion, associated to plastic deformation of the coating under the pressure of the tip.
Comparison with values obtained for catechol 1-terminated SAMs shows that a) the average adhesion of the catechol monolayer is slightly higher than the maximum adhesion force recorded on the PDA thin film, and b) much more consistent results are obtained on SAMs owing to their intrinsically homogenous nature (both chemical and spatial). to an increase in the distance between neighbouring catechol tail groups as surface roughness increases, [28] leading to the deposition of a poorly packed monolayer, and consequently a decrease in the adhesion force. Moreover, the rougher topography of the substrate is expected to lead to important variations in the contact geometry between the tip and the sample, thus adding uncertainty to the results. As an alternative to AFM measurements, the same coated substrate was immersed in a colloidal solution of iron oxide nanoparticles (Ø ~ 8-10 nm) and sonicated for 15 min. [13a] A relatively homogenous distribution of nanoparticles was observed across the whole surface, as shown in Figure 3. This procedure was repeated for comparison purposes on two additional polycrystalline gold surfaces; namely, an unmodified, bare substrate and a polydopamine-coated substrate. Very few nanoparticles were found randomly adsorbed on bare gold substrates, while the same experiment on the polydopamine-coated samples was not conclusive due to damage of the coating upon sonication (see Supporting Information, Figure S5). Therefore, despite the fact that roughness of the substrate impedes a precise assessment of the monolayer adhesion by AFM, the thiols seem to be still homogeneously distributed on catechol-coated surfaces, with enough catechol groups available for NP attachment.

Monolayer Formation Process
The alkanethiol adsorption and equilibration time needed for the formations of SAMs has been thoroughly studied and is assumed to occur in a two-step mechanism influenced by the chemical nature of the head groups. [29] However, the vast majority of these studies are based on long chain alkanethiols, whose chemical nature is only roughly comparable to compound 1.  Figure S5), far below those found for catechol-terminated SAMs with longer immersion times, and otherwise rather similar to those obtained for long alkyl chains. Following a previous approximation, a polycrystalline gold substrate that had been kept in the thiol solution for a short period of time (15 min) was then immersed in a dispersion of iron oxide nanoparticles. The particle coverage obtained on this substrate was shown to be poor and inhomogeneous. Overall, these results would suggest that transient layers (i.e. before equilibrium) afford surfaces with essentially non-adhesive character.
Confirmation for the need of having sufficiently prolonged immersion times to obtain good quality monolayers was obtained by fabricating monolayer dot arrays with a direct-write scanning probe lithography technique such as Dip-Pen Nanolithography (DPN). [30] This methodology has been previously shown to form close-packed and highly ordered SAMs with commonly used thiols such as ODT and mercaptohexadecanoic acid provided that appropriate deposition and solvent evaporation conditions are chosen. [31] Contact mode AFM images obtained immediately after the deposition using the same coated tip showed a difference in friction on the spots where the dip pen deposition procedure was performed, proving that the catechol-terminated thiol had efficiently transferred to the surface (Figure 4a).
Arrays of deposited catechol 1 were then located by LFM imaging using a clean tip (Figure   4b). F-d curves were recorded afterwards on both functionalised and pristine areas while scanning the surface with the same probe. No significant differences were observed between the adhesion values of bare gold and the coated areas, showing that droplet evaporation takes places before the equilibrium conditions for the SAM formation are achieved. These results were reproducible for additional square motifs obtained with slow writing speeds and repeated passes, which should have contributed to improve the monolayer quality. It is important to point out here that at least one preliminary scan of the area is required to locate the arrays before measuring the adhesion, meaning that both the surface and the tip can be modified in this process thus affecting the subsequent measurements. [32] Nevertheless, in view of these results we can conclude that no significant enhanced adhesion could be measured on sub-monolayers prepared by DPN, suggesting that this technique affords poor-quality monolayers of 1.
-Insert Figure 4 here-In order to get some atomistic insight into the formation of the studied monolayers, all-atomic Molecular Dynamics (MD) simulations were carried out (see Simulation Methods Section).
Atomically-flat gold surfaces with different coverage degrees of 1 were considered, both in vacuo and with water as solvent. All MD simulations were performed at 25ºC and, wherever present in the simulation, the solvent was kept at 1 atm of pressure. The spatial organization of compound 1 with regard to the surface was characterized by measuring the tilt angle α, the dependence of which with molecular coverage is shown in Figure 5a. At very low values (< 1 molec/nm 2 ), molecules of 1 tend to lie roughly flat on the surface (α≈0º), with catechol groups adsorbed at the interface, as would be expected for a monolayer in its first formation stages (Figure 5b). Higher surface coverages of 1 generate equilibrium configurations with raising tilt angles, so that at about 2 molec/nm 2 , catechol groups appear substantially desorbed from the gold surface with α≈30º. This trend continues at least up to a surface coverage of ca.
3.6 molecules/nm 2 -the largest coverage simulated-, for which a tilt value of α≈60º is obtained.
As can be seen, the effect of the solvent is not very important except at large coverage values, when it tends to induce larger tilts as compared to the in vacuo case.
During the simulations, we have also computed the energy per molecule -the sum of the interactions with other molecules, solvent and surface, plus the conformational energy, and the kinetic energy due to thermal agitation-for each surface coverage value, and hence for each estimated tilt angle. For SAMs in presence of water (Figure 5a), and very low coverages (<1 molec/nm 2 ) the molecular energy is found to be roughly constant and consistent with a sparse coverage of independent, randomly oriented and flat-lying molecules (Figure 5b). For higher surface coverages, more favourable intermolecular interactions, bring about a consistent decrease in the molecular energy, concomitant to increasing tilt angles (Figure 5c), suggesting that the spontaneous formation of monolayers of 1 should be energetically favoured. According to the MD calculations, the energy seems to reach a minimum at a coverage of about 3.33 molecules/nm 2 (corresponding to a tilt angle of α≈60º ), meaning this would be the energetically preferred coverage for SAMs made of compound 1. The energy per molecule increases for larger coverages, which is attributable to packing constraints and steric interactions between adjacent molecules.

Summary
The spectroscopic characterization (PM-IRRAS, XPS, ellipsometry) of gold substrates coated with catechol-thiol 1 showed the tendency of this molecule to self-assemble on gold surfaces forming monolayers with outward-facing catechol groups. For this, sufficiently prolonged immersion times (i.e. longer than for long-chain alkanethiols) of atomically flat gold surfaces in solutions of 1 were mandatory in order to obtain good quality monolayers. The spontaneous formation of monolayers of 1 on gold was supported by theoretical calculations showing that this process should be energetically favoured.
With regard to the average adhesion force of catechol-terminated monolayers at the molecular level, it was found to be five times higher than that of bare gold, and eight times higher than that of an ODT monolayer. Comparison with PDA coated substrates also support the existence of enhanced adhesion for the monolayers, as F-d curves measured on PDA coatings not only showed overall lower adhesion than those of catechol 1 SAMs, but much lower reproducibility as well, hinting at their inherent lack of structural and chemical homogeneity.
Finally, surface roughness was also shown to impair the final adhesion properties of the monolayer: an increase on the surface roughness led to a severely diminished adhesion force at the molecular scale, although coated 'rough' substrates still showed the ability to organize magnetic nanoparticles on its surface.

Synthesis. General Procedures.
Commercially available reagents were used as received. Epitaxial gold (300 nm) on mica substrates were purchased from Georg Albert PVD and stored under vacuum. Prior to the SAM formation, the epitaxial gold substrates were cleaned by carefully rinsing with acetone, EtOH and Milli-Q water and dried under a nitrogen stream.

SAM formation.
All the substrates were cleaned in a UV/O 3 cleaner for 10 min (Novascan Technologies) and immediately immersed in the corresponding solutions. SAM formation was obtained by a standard procedure, as follows. Clean gold substrates were immersed overnight in 1 mM solutions of the corresponding thiols in EtOH. Then, the substrates were rinsed with copious amounts of EtOH and Milli-Q water and dried by a nitrogen flow. For the force-distance measurements, the modified substrates were allowed to dry overnight; for the rest of the experiments, they were immediately used as prepared.

SAM characterization.
Polarization modulation infrared reflection-absorption spectra were recorded on a FT-IR spectrometer Vertex 70(Bruker) combined with a PMA50 accessory. The angle of incidence during the acquisition of the spectra was 80º. Two separated spectra were recorded with the photoelastic modulator set at 2900 cm -1 for the OH and CH 2 stretching region and at 1600 cm -1 for C=C and CO stretching and OH bending region. X-ray photoelectron spectroscopy was carried out in a Phoibos 150 analyzer (SPECS GmbH, Berlin,Germany)) in ultra-high vacuum conditions (base pressure 1·10 -10 mbar). A monochromatic Kα X-ray source (1486.6eV) was used. The spectra were based on photoelectrons with a takeoff angle of 0º for the S 2p core level and 30º for the C 1s core level (takeoff angle considered with respect to the surface normal).
Sprectroscopic Ellipsometry was carried out in a Semilab Sopra GES5E spectroscopic ellipsometer in order to determine the thickness and refractive index of the layered structures.
During the investigation the incident angle of the light beam was set to 65, 70 or 75 degrees and the wavelength was varied from 300 to 800 nm. Measurements were carried out at least three macroscopically spaced points on the sample. Ellipsometric data was fitted with multilayer models using the Semilab's WinElli II analysis software. Clean gold substrates were modelled first. Next, a Cauchy model (A = 1.49) was used to model the organic film.

Polydopamine synthesis and surface coating.
Polydopamine was obtained following the previously described procedure. [1] Dopamine hydrochloride (2 mg/mL) was dissolved in 10 mM Tris·HCl (pH 8.6) solution. The substrates were placed in vertical orientation into the mixture solution and for 1 hour while stirring to minimize non-specific deposition. Afterwards, the substrates were rinsed with Milli-Q water and dried under a nitrogen stream.

Iron oxide nanoparticles: synthesis and deposition.
Maghemite nanoparticles were obtained by coprecipitation.  (1) in ACN for about 10 seconds and dried with gentle nitrogen flow. After that, the tip was exposed to water vapor for 5 min and then left to dry. Once dry, the tip was re-dipped again in the same solution and blow-dried with nitrogen. It is worth noting that epitaxial gold surfaces are quite fragile and soft, so careful adjust of the parameters of the processes, such as laser alignment and deflection setpoint, was required when performing the lithographies and subsequent characterization steps to avoid indentation of the surface.

Molecular Dynamics Simulations.
Molecular Dynamics (MD) simulations are based on the numerical solution of the Newtonian equations of motion for all atoms of a molecular system constrained to the given thermodynamic conditions. All MD simulations were performed using the NAMD software, [33]