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New Concepts at the Interface: Novel Viewpoints and Interpretations, Theory and Computations
Reversible Self-Assembled Monolayers (rSAMs) as Robust and Fluidic Lipid Bilayer Mimics Sing Yee Yeung, Thomas Ederth, Guoqing Pan, Judita Cic#nait#, Marite Cardenas, Thomas Arnebrant, and Börje Sellergren Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00226 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018
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Reversible Self-Assembled Monolayers (rSAMs) as Robust and Fluidic Lipid Bilayer Mimics Sing Yee Yeunga, Thomas Ederthb, Guoqing Pana, Judita Cicėnaitėa, Marité Cárdenasa, Thomas Arnebranta and Börje Sellergrena* a
Department of Biomedical Sciences and Biofilms-Research Center for Biointerfaces (BRCB), Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden.
b
Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, 581 83 Linköping, Sweden
Abstract Lipid bilayers, forming the outer barrier of cells, display a wide array of proteins and carbohydrates for modulating interfacial biological interactions. Formed by the spontaneous selfassembly of lipid molecules, these bilayers feature liquid crystalline order, while retaining a high degree of lateral mobility. Studies of these dynamic phenomena have been hampered by the fragility and instability of corresponding biomimetic cell membrane models. Here, we present the construct of a series of oligoethylene glycol-terminated reversible self-assembled monolayers (rSAMs) featuring lipid bilayer-like fluidity, while retaining air and protein stability and resistance. These robust and ordered layers were prepared by simply immersing a carboxylic acidterminated self-assembled monolayer into 5-50 µM aqueous ω-(ethylene glycol),α-(4amidinophenoxy)decane solutions. It is anticipated that this new class of robust and fluidic twodimensional biomimetic surfaces will impact the design of rugged cell surface mimics and highperformance biosensors. 1 ACS Paragon Plus Environment
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Introduction The surface of eukaryotic cells is covered by a dense layer of proteins and carbohydrates. Presented towards the extracellular environment, they act as cellular guards and barcodes modulating biological activities through interactions with extracellular receptors or ligands.1-2 Separating the intracellular components from the external environment is a 3-5 nm thick lipid bilayer membrane. One of its unique properties is the ability to retain liquid crystalline order, while demonstrating two-dimensional fluidity. This feature holds key information in regulating affinity and selectivity of biological interfacial interactions by dynamically controlling the spatial arrangement and orientation of proteins and carbohydrates. In order to decipher these phenomena, researchers make use of simplified cell membrane models.3-4 The most well-studied two-dimensional biomimetic cellular membrane models on solid supports are self-assembled monolayers (SAMs)
5-9
and supported lipid bilayers (SLBs).10-13 The former
have the advantage of control over ligand density, homogeneity and orientation, allowing unambiguous interaction studies. However, it lacks lateral mobility, which is one of the most important aspects of cellular membranes.14 SLBs are laterally mobile but they are not robust enough to be used as biosensors. The layers formed are often not stable in air and prone to exchange with proteins. Air-stable and robust alternatives such as hybrid lipid layers often lose their lateral mobility.10, 15-17 Literature examples that contain both characteristics are rare and typically require extensive laboratory skills to fabricate.18-21 We have recently reported on an adaptable biosensing platform, reversible self-assembled monolayers (rSAMs), featuring strongly enhanced affinity and sensitivity.22 This new sensing system utilizes noncovalent amidinium-carboxylate ion pairs for building up stable two-dimensional assemblies, akin to lipid bilayers but with a simple preparation process, enhanced rinsing stability 2 ACS Paragon Plus Environment
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and fast on/off rates (Figure 1A). These benzamidine-terminated amphiphiles spontaneously assemble in neutral or alkaline aqueous solution on alkanoic acid functionalized thiol SAMs to form ordered layers with tunable pH responsiveness. Layer thicknesses and order correlate with the molecular length of the amphiphile. When the alkyl chain exceeds a certain length, the rSAMs feature crystalline-like order and an odd-even chain length-related tendency to form bilayers.23-25 For practical applications as cell membrane mimics and biosensors, these surfaces have to be stable towards modification at the ω-position with hydrophilic ligands and show resistance towards non-specific interactions and protein exchange. Attachment of ethylene glycol repeating units at the terminal end is one of the most common and well-studied approaches to impart protein resistance and to provide spacing for ligand attachment.26-27 However, the self-assembly process of amphiphilic molecules in aqueous media is governed by the balance between attractive hydrophobic and repulsive steric or electrostatic interactions in the hydrophilic parts.28-29 The increased steric repulsive force with the introduction of ethylene glycol units at the ω-position would challenge the amphiphiles’ ability to spontaneously self-assemble into ordered domains, thereby compromising the stability and resistance of the surfaces towards protein exchange. To address these issues we have synthesized a series of α-(4-amidinophenoxy)decanes decorated at the ω-position with even repeating units of ethylene glycol, E0-6 (Scheme S1) and evaluated their ability to assemble into well-ordered layers via in situ and ex situ ellipsometry, infrared reflection-absorption spectroscopy (IRAS) and atomic force microscopy (AFM) (Figure 1). Immobilization parameters were systematically examined to optimize stability and molecular order at close to physiological conditions. The formed layers displayed a lipid bilayer like fluidity and pH-induced switchability, but were stable towards air and protein exposure and rinsing in buffer. 3 ACS Paragon Plus Environment
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Figure 1. Formation of reversible self-assembled monolayers (rSAMs). A Schematic illustration of the preparation of ethylene glycol (E0-6) terminated rSAMs on 16-mercaptohecadecanoic acid (MHA) or 4-mercaptobenzoic acid (MBA) self-assembled monolayers (SAMs).22-25 B Real time in situ ellipsometric thickness of rSAMs featuring two repeat units of ethylene glycol, E2 on MHA-SAMs demonstrating pH responsiveness and reversibility.
Results and Discussion
A series of ω-(ethylene glycol)n-α-(4-amidinophenoxy)decanes, (n = 0-6, E0-6) were synthesized according to Scheme S1. The assembly kinetics and rinse stability of E0-6 were first evaluated using in situ ellipsometry at pH 9 borate buffer followed by pH 8 HEPES buffer rinsing as previ4 ACS Paragon Plus Environment
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ously reported.22 Using IRAS and AFM, further information regarding the molecular order of the formed layers were subsequently obtained.
Formation and surface characteristics of E0-6 rSAMs on MHA-SAMs. The rSAM film thickness on MHA-SAMs were measured in real time upon exposure to 50 µM E0-6 in pH 9 borate buffer solution (Figure 2A). The rate of adsorption of amphiphiles E0-E4 increase with the number of ethylene glycol units, with E0, featuring only the terminal hydroxyethyl functionality, adsorbing distinctively slower than the other two ethylene glycol-tethered amphiphiles. Equilibration of the surfaces with the amphiphilic solutions gave the limiting equilibrium thicknesses, Dads shown in Figure 2B. E0-E4 were stable and formed layers with thickness exceeding the calculated length of the amphiphiles. As we recently concluded, this agrees with the formation of bilayered assemblies.22 The behavior of E0-E4 contrasted with E6 that spontaneously slowly desorbed after the initial adsorption phase. The enhanced water solubility of the ethylene glycol-terminated amphiphiles implies that they are present predominantly in monomeric form and can rapidly diffuse to the surface.28 In contrast, E0 is poorly water soluble and thus likely to adsorb in the form of aggregates.22 Hydrophilic head groups will also contribute to a lowering of the surface tension but may on the other hand be more sterically demanding, compromising rSAM close packing. Indeed, for surfaces that formed stable layers (E0-E4), the equilibrium thickness, Dads correlated inversely with the calculated length of its corresponding amphiphiles. This indicates that steric repulsion from the ethylene glycol addition prevents close packing or bilayer formation. The odd behavior of E6 can be explained
by
the
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Figure 2. Characterization of E0-6 rSAMs on MHA-SAMs. A Real time in situ ellipsometric thickness of MHA-SAMs upon exposure to E0-6 (50 µM, pH 9 borate buffer). B In situ ellipsometric thickness after equilibration, Dads and after pH 8 HEPES buffer rinsing, Drinse. For E6, only layers with stable equilibrium thicknesses were included in the calculations. C Baseline corrected high (top) and low (bottom) frequency regions of E2 bulk ATR spectra (black traces) and E2 modified MHA-SAMs IRAS spectra (red traces) D Atomic force microscopy (AFM) topographic images and cross sectional profile of E2 modified MHA-SAMs. Details of E0-6 rSAMs characterizations are summarized in Table S1. 6 ACS Paragon Plus Environment
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presence of two competing processes distinguished by different kinetics. Possibly, a fast surface assembly is here competing with a slower formation of a thermodynamically more stable supramolecular assembly (e.g. micelle).30
The surfaces were then rinsed in situ in a continuous system with pH 8 HEPES buffer to investigate their stability. The thickness of the film remaining after rinsing the layers, Drinse is inversely correlated with the ethylene glycol chain length (Figure 2B and Table S1). Nevertheless, Drinse of layers E0-4 are still larger than the calculated length of the molecules, indicating the presence of stable monolayers or partial bilayered assemblies (vide supra).
To gain further insight into the layers’ molecular order and orientation, the E2 and E4 rSAMs were dried under a nitrogen stream and the IRAS spectra were collected (Figure 2C and Figure S1). Comparing the layer IRAS spectrum with the bulk ATR spectrum of E2, the layer spectrum exhibits different relative band intensities and bandwidths (for detailed peak assignments see Table S2). In the high frequency region of the E2 rSAM spectrum, the CH2 stretch vibrations at 2918 cm-1 (asym) and 2850 cm-1 (sym) and the sharpness of these bands of the layer spectra indicate the presence of all trans extended chains of closely packed amphiphiles. The pronounced increase of (C=C)1,4 at ca. 1611 cm-1 and concomitant decrease of aromatic C-H out-of-plane bending mode at 840 cm-1 suggests a near upright position of the amphiphile assemblies. Taking the peak intensity ratio of the layer and bulk spectra of aromatic C-H out-of-plane bending mode at ca. 840 cm-1 and (C=C)1,4 at 1611 cm-1, the phenyl group of the amphiphiles are determined to have a tilt angle of ca. 18-20° relative to the surface normal.23
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To verify the results, each surface was sampled twice at different areas and the experiments were duplicated on a separate substrate (Table S1). The average CH2 asym and sym stretch vibrations decrease in wavenumber with increasing number of ethylene glycol repeating units (E0>E2>E4). This contradicts the general consensus that oligoethylene glycol causes steric repulsion and reduces molecular order of SAMs containing increasing number of E repeat units.28-29,
31-32
As
IRAS is an averaging technique, these results can also be attributed to the interference from a loosely packed 2nd layer.
The ellipsometric thicknesses reported in Figure 2B suggests a decrease in top layer coverage with increasing ethylene glycol chain length (E4
