ABSTRACT
Another popular and powerful surface modification strategy is self-assembly: it is easy to apply, which makes it possible to change or tune the surface character by modifying the end groups of the layer accurately and predictably. The self-assembly strategy, which forms surfaces with stimuli-responsive properties, is also known as smart surfaces and is discussed in the following section. 3.3.1 Thiol-Gold Coordination: Self-Assembled
Monolayers (SAMs)The process of self-assembly involves the spontaneous non-covalent arrangements of atoms and molecules to form a functional unity toward an energetically stable form, whose novel structure and properties are determined by their nature.24 Natural and prominent examples of self-assembly are provided by double-helical25 and triple-helical DNA,26 multichromophore structures of the photosynthetic reaction center,27 and light-harvesting antennae apparatus.28 Among self-assembly strategies, the generation of a self-assembled monolayer (SAM) is one of the most elegant ways to create a film with specific surface properties.4A self-assembled monolayer is a layer of molecular thickness formed by self-organization of active surfactant molecules in an ordered manner on a solid surface. Molecular structures forming SAMs can be divided into three parts (Fig. 3.1):29-31
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Figure 3.1 Model for self-assembled monolayer of alkanethiols on gold. • A surface-active head group, which strongly binds to the solid substrate. • An end group that allows the introduction of a variety of organic functionalities (e.g., acidic group, ester, amide, or alcohol) and plays an important role in terms of coupling the biomolecule to a monolayer. • A spacer unit (alkyl chain) that connects the head group and end group, giving stability to the assembly by van der Waals interactions, affecting the intermolecular separation and molecular orientation in the film, while ensuring that the reactive groups targeting the analytes are far enough from the surface so as to reduce any putative deleterious influence. Each those constituents can be easily manipulated and controlled by various chemistries, making them compatible for desired applications. Through proper selection of terminal functional
groups, specific surface interactions can be exploited to immobilize molecules at an interface. The most common strategy is based on the use of alkanethiol molecules, which attach strongly to gold surfaces via their sulfur atoms, forming SAMs. In fact, this forms good, well-packed SAMs and is historically the most studied strategy. In general, sulfur compounds are well known for their reactivity to noble metals. Gold surfaces offer an advantage of being easily functionalized with SAMs and have been extensively used, although a variety of other substrates were possible candidates for the
deposition of sulfur moieties.32 Because the formation of SAMs on such surfaces depends on the crystalline morphology of the metal, gold yields SAMs with the highest density and degree of regularity.19In addition, the method to modify gold surfaces with self-assembled thiol derivatives is extremely simple and can be achieved in a laboratory just by immersing the desired slides into a thiol solution for a specified time followed by thorough washing with the same solvent and drying, often using a jet of dry argon.33SAM formation, pioneered by Nuzzo34 in 1983, demonstrated that dialkyl disulfides generate oriented monolayers on gold. This opened a new era for immobilization, and many studies have been performed since then, showing that alkanethiols adsorb from solution onto gold, forming S-Au bonds.35-36 This bond forms spontaneously, when the thiol end of the alkanethiol approaches the gold, resulting in the weakening of the sulfur-hydrogen bond and release of hydrogen (Fig. 3.1):37-40 R-SH + A u n0 → R-S-Au+ ∙ A u n0 + 1/2 H2The thiolate molecule acts as nucleophile and donates a pair of electrons to the gold surface, forming a strong covalent bond. Despite its zero valence, inert gold binds sulfur with longer Au-S bonds than Au-Au, demonstrating a weak but partial covalent bonding with –H the leaving group, which tends to combine into H2 molecules.41,42Long chain alkanethiols (X(CH2)nSH, n = 11 – 18) connect to a gold surface by forming densely packed, ordered, oriented monolayer films and are considered to be thermally more stable than films formed from short chain thiols.43 Such crystalline-like structures, with all-trans conformation alkyl chains, exhibit average tilt angles of the chain axis in a range of 28° to 40° from the surface normal, and an approximate 55° twist (rotation angle) of the chain axis away from a configuration with the plane perpendicular to the surface.44,45 The dense packing of SAMs results mainly from van der Waals interactions between the neighboring carbon chains.31 The separation distance between neighboring alkanethiols is 0.5 nm, representing a ( √__ 3 × √__ 3) R 30o overlayer structure on the gold surface.31,46-48 In order to stabilize the SAMs even more, internal substitutions can be performed. It has been reported that increasing the stability of such a layer can be achieved by incorporation of
internal hydrogen bonding cross-links, such as amide-containing chains instead of methylene chains.49,50 Because the resulting hydrogen bond between the amide groups of neighboring chains are stronger than van der Waals interactions, it will deform at higher temperatures.51 Porter et al.52 have demonstrated the importance of the alkyl chain length on the packing density, intermolecular environment, and geometry of the monomolecular assemblies, stating that with a decrease of the chain length (n < 8), the layer becomes increasingly disordered. Therefore, chains containing mainly n > 10 carbons can form well-organized structures. Another property affecting the packing and molecular orientation in SAMs is the type of terminating groups and their mutual
interactions. It has been reported that differently functionalized end groups of n-alkanethiols (COOH-, CH3-, and OH-terminated thiols) with the same chain length will not form the same packing, thus influencing the structure of alkanethiolate monolayers on gold substrates.53 The crystalline-like monolayer can be destabilized by different environmental conditions, such as elevated temperature or extreme pH.54,55 Other factors influencing the fabrication of SAMs are the nature of the solvent and the time of immersion of gold surfaces in concentrated thiol solutions, while longer periods may result in the formation of multilayers.56All the aforementioned intrinsic properties of alkanethiol-based SAMs on a golden surface enable reliable construction of sensing systems. The stability, the uniform surface structure, and the relative ease of their functional modification make SAMs an ideal choice for immobilizing sensing molecules on surface. The design and use of SAMs for application in biomolecular recognition, including different kinds of modifications and their dynamic interactions with biomolecules, are reviewed in the following sections. 3.3.1.1 Monothiol layersThe vast majority of alkanethiols derivatives contain a single –SH group and are anchored to gold via single S-Au bonds. These molecules differ by their size and by the functional terminating group for attachment of biomolecules, as discussed above. The functional terminal group can be acidic (COOH-terminated thiols), amide
(NH2-terminated thiols), alcohol (OH-terminated thiols), or phosphonic (–PO3H2). 3.3.1.1.1 Alkanethiol with carboxylic terminus (–COOH)The most popular molecules for immobilization are alkanethiols with a carboxylic acid tail, varying by chain size. In comparison to their simple alkanethiol analogues, the organization of acid SAMs is complicated by the presence of the carboxyl head groups. When protonated, strong hydrogen bonding between the –COOH head groups can influence the functionality of the film, whereas in the deprotonated state, electrostatic repulsions between neighboring carboxylate anions can disrupt the film’s packing density. Therefore, both existing and future applications of these surfaces rely on a thorough understanding of the state of the carboxylic acid group from which further surface functionality can be derived.57The molecule with the shortest chain is a thioglycolic acid (Fig. 3.2y), comprising two carbons, one attached to –COOH for bio-conjugation and the other to –SH, to form a S-Au bond with gold.58-60The most common chain length is 11-mercaptoundecanoic acid (11-MUA) (Fig. 3.2q),61,62 16-mercaptohexadecanoic acid (MHDA) (Fig. 3.2l),63 and 3-mercaptopropionic acid (MPA) (Fig. 3.2o).64,65The popularity of the carboxylic-terminated molecules is mainly because of the relatively simple protocol needed to modify them for biomolecule immobilization, usually with ethyl-3-(3-dimethyl-ami-no-propyl)-carbodiimide (EDC) and N-hydroxy-succinimide (NHS). Those long-chain alkanethiols form highly ordered and densely packed monolayers suitable for bioconjugation.4,36 Nevertheless, other studies show that the –COOH terminus of the chains form strong hydrogen bonds with other –COOH groups leading to a dimerization of the COOH groups, thus preventing the formation of highly oriented films.53 Such dimerization can be prevented by adding acetic acid to the thiol solution, which will protonate the COO-tails.66 The monolayer can achieve a significantly higher degree of order by using a more rigid backbone chain, consisting of aromatic rings such as mercaptohexadecanoic acid and mercapto-methyl-terphenyl-carboxylic acid, instead of a CH2 backbone.67
