Biophysics at the Nanoscale: Applications of Functional Materials

Approx.230 pages

Approx.230 pages

• Language: English
• Publisher: Academic Press
• Release date: Oct 8, 2023
• ISBN: 9780443153600

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Chapter 1

Self-assembled monolayer–based nanoscaled surfaces

Faezeh Ghorbanizamani¹*, Emine Guler Celik²*, Hichem Moulahoum¹ and Suna Timur¹, ³,    ¹Department of Biochemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey,    ²Department of Bioengineering, Faculty of Engineering, Ege University, Bornova, Izmir, Turkey,    ³Central Research Test and Analysis Laboratory Application and Research Center, Ege University, Bornova, Izmir, Turkey

Abstract

Self-assembled monolayers (SAMs) are naturally occurring self-organized structures or biologically inspired engineered architectures exhibiting well-oriented, chemically functional, stable, flexible, and micro- and nanoscaled surfaces. The design and integration of SAMs for building different materials, systems, and devices have shown rapid progress in the fields of biosensors and nanotechnology. The combination of the structural and dynamic properties of SAMs with biosensors provides highly sensitive, miniaturized, integrated, and high-performance analytical devices. Additionally, SAMs give versatile two- and three-dimensional assemblies, which can be further modified with functional chemical groups and biomolecular units for the desired application (e.g., regenerative medicine, tissue engineering, drug delivery, biosensors, electronic devices, and transistors). This chapter outlines the structural properties and various applications of thiolate-, polymer-, dendron-, lipid-, and peptide-based SAMs. Finally, an overview of recent studies was given to understand the critical role of SAMs in forming nanometer-sized structures with functional, biocompatible, well-defined, controllable, stable, and flexible properties.

Keywords

Self-assembled monolayers (SAMs); nanoscaled surfaces; thiolate SAMs; dendron SAMs; polymer SAMs; lipid SAMs; peptide SAMs

1.1 Self-assembled monolayers

The ability of organic thiols to produce a self-assembled monolayer (SAM) on gold surfaces was first discovered by Nuzzo and Allara in 1983 [1]. Such a discovery opened a new window for tailoring and fictionalizing different surfaces at the molecular level [2–4]. These systems enable precise control of physical and chemical surface properties, which expand their applications in various fields of nanotechnology and energy industries, for example, chemicals and biosensors [5–7], biotechnology [8,9], molecular electronic devices [10–12], solar cells [13], electrochemistry, and organic field-effect transistors [14]. SAMs are super-organized thin films naturally arranged on certain substrates by physical phenomena, including electrostatic and hydrogen-bonding interactions. SAMs’ constructive molecules are composed of a chemical head assembly that attaches to the substrate and a tail group that can be used to control the film’s surface properties. The lateral conjugation of adjacent constructive molecules leads to the formation of densely packed, oriented monomolecular films (Fig. 1.1A). The features of SAMs could be wisely tuned by selecting their constructive molecules. The most explored SAMs are composed of alkanethiols and disulfides on gold and silver underlying substrates or alkylsiloxanes on silica, glass, and alumina surfaces that consist of hydroxyl groups [1,15–17]. The most recently created SAMs have been developed by applying amphiphilic block copolymers, which use hydrophobic interactions to form SAM-like layers on the polymeric surface [18,19].

Figure 1.1 (A) Preparation of a self-assembled monolayer molecule [20]. (B) Representation of a thiol molecule highlighting the head group, spacer, and tail group. (C) Self-assembly of alkanethiol on gold surfaces [21]. (D) Reactions for the preparation of self-assembled monolayer over gold surfaces. Formation of trichlorosilane±SiO2 and gold±thiol monolayers (upper reaction). The carboxyl-terminated film activation with ethyl chloroformate, thionyl chloride, etc. for amine or alcohol replacements (middle reaction). The carboxyl-terminated film activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for amine replacement (bottom reaction). (A) Inspired from M.A. Reed, J.M. Tour, Computing with molecules, Sci. Am. 282(6) (2000) 86–93; (C) Reproduced with permission from C. Vericat, M.E. Vela, G. Benitez, P. Carro, R.C. Salvarezza, Self-assembled monolayers of thiols and dithiols on gold: new challenges for a well-known system, Chem. Soc. Rev. 39(5) (2010) 1805–1834. © 2010 Royal Society of Chemistry.

The significant growth in SAM research and application has demonstrated the need for flexible design of two- and three-dimensional assemblies at the individual molecular and material levels. Here, we describe different SAMs, their exclusive properties, and their application in various fields.

1.2 Self-assembled monolayers based on thiolates

The alkanethiols monolayer spontaneously assembled on noble metals is the most studied and considered SAM system. The sulfur head group creates strong interactions with the noble metals’ surface (specifically gold). The thiolates’ self-assembly on noble metal surfaces is driven by inter- and intramolecular forces (electrostatic and hydrophobic interactions, hydrogen bonds, and van der Waals forces). The sulfur group has a binding affinity for metals via semicovalent bonds. The calculated energy for sulfur–gold interaction (45 kcal/mol) shows the formation of more stable bands in comparison with the C—C bond (~83 kcal/mol) [22]. Other than the affinity force, the hydrophobic and van der Waals interactions generated by alkane or aromatic chains could drive the formation of the self-assembled layer. These driving forces tilt the thiol chains to expand the interaction between the chains and reduce the overall surface energy. The carbon-based chain could control the properties of the whole structure and the well-ordered SAM platforms. Thus, a typical SAM platform based on thiolates consists of three main constructive parts: (1) a sulfur head group that attaches to a noble metal surface, (2) an alkane chain (typically 10–18 carbons in length) or aromatic chain, and (3) a tail (or functional terminal group) allowing many functional groups (e.g., halogens, hydroxyls, carboxylic acids, amides, etc.). The sulfur head groups can strongly bind to metals such as gold, silver, copper, platinum, palladium, and nickel [23]. Gold (Au) could attract the most attention among all these metals due to its physicochemical stability, surface chemistry, biocompatibility, and optical features. As gold resists oxidation, its flat and clean surface can be directly modified by thiols without any pretreatment in gas or liquid phases under moderate and ambient conditions [21]. In liquid phases, adsorption is generally accomplished depending on the molecule’s nature (0.01–1.0 M thiol concentration). To have a well-ordered SAM surface, the adsorption time could be changed from 2 to 24 hours based on the length and features of thiol molecules [6,24].

On the other hand, the creation of thiolate–Au SAM through gas-phase deposition is a complex procedure requiring different steps (Fig. 1.2), starting from physisorption, then chemisorption, and finally, the crystalline formation that produces organized domains with molecules configured in a closed-packed structure [21]. However, one of the advantages of thiolate–Au SAM is the alkanethiols’ ability to physically and chemically adsorb via van der Waals interactions and the sulfur head group, respectively. Taking advantage of Au features, thiolate SAMs on gold have been utilized in various applications in specific biological systems, such as fluorescent biological detection of pathogens and proteins, immunoassays, biosensors, and optical-light responsive tools. Table 1.1 summarizes some of the thiolate–Au SAM design approaches as biosensors. Moreover, the formation of gold–thiolate on a metallic core creates a template that could be employed in biology, medicine (therapy, diagnostics, and imaging), catalysis, photonics, and electronics [25–28]. In addition to SAMs’ structure, the chemistry of the tail group plays a critical role in using thiolate–Au SAMs in different applications. Terminal carbonyl groups in monolayers could be formed by the addition of carbodiimides (dicyclohexyl carbodiimide (DCC) or 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)) [29]. Carboxyl-terminated films could be used to form anhydride terminal groups through the reaction with ethyl chloroformate [29]. Moreover, the surface exposure of carboxyl-terminated films to gaseous SOCl2 was shown to yield carboxyl chloride groups [29]. These activated derivatives can produce esters or amides due to their easy reactions with alcohols or amines (Fig. 1.1D). The direct deposition of thiol/disulfide or EDC-mediated coupling of acid-terminated films with N-hydroxysulfosuccinimide leads to the formation of ester-functionalized films [29]. These structures can be employed to attach amines with high yields. Monolayers with terminal amine groups could also be used to react with acylating reagents such as acid chlorides [30], active esters [31,32], or quinones [33]. In addition, esters are also produced through the acylation of hydroxyl groups found in monolayers by acid chlorides and anhydrides [34].

Figure 1.2 (A) Small molecule-based self-assembled monolayers (SAMs) and polymeric-based SAMs (PSAMs). (B) Grafting models of different copolymers with different active groups. (C) PSAMs structures obtained through (a) bulk solution phases [35]. (b) thin film [36], or (c) substrate-patterned surfaces. (B) Reproduced with permission from J.W. Park, H. Kim, M. Han, Polymeric self-assembled monolayers derived from surface-active copolymers: a modular approach to functionalized surfaces, Chem. Soc. Rev. 39(8) (2010) 2935–2947 © 2010 Royal Society of Chemistry. (C) (a) Reprinted with permission from S. Forster, T. Plantenberg, From self-organizing polymers to nanohybrid and biomaterials, Angew. Chem. Int. Ed. Engl. 41(5) (2002) 689–714. © 2002 WILEY-VCH Verlag GmbH; (b) reproduced with permission from R.A. Segalman, Patterning with block copolymer thin films, Mat. Sci. Eng. R. 48(6) (2005) 191–226. © 2005 Elsevier B.V.; (c) reproduced with permission from J.W. Park, H. Kim, M. Han, Polymeric self-assembled monolayers derived from surface-active copolymers: a modular approach to functionalized surfaces, Chem. Soc. Rev. 39(8) (2010) 2935–2947 © 2010 Royal Society of Chemistry.

Table 1.1

1.3 Self-assembled monolayers based on polymers

Typical SAMs consist of small amphiphilic molecules with head and tail groups responsible for attaching to the solid substrate surface and target molecules. Other than small molecules, macromolecules such as polymers could be implicated in SAMs’ preparation (PSAMs). The long macromolecule chain in PSAMs can be attached to the surface by employing active sites on the end or side of the chain by forming single or multiple bonds (Fig. 1.2A) [53]. Polymers with active sites in the entire chain length can be adsorbed parallelly on the surface. On the contrary, polymers with surface-active sites in specific parts of repeating units could be grafted on the surface either randomly or using block side and end side grafting. For copolymers with a backbone containing sticky block reactive units, the adsorption happens parallel to the surface. In contrast, the rest of the block is grafted on the side and can change conformation freely. The assemblies in which one end of the chain is tethered to a surface are known as polymer brushes. The modified polymer brushes with various materials (glass, gold, silver, titanium) have been used for a variety of medical applications (tissue engineering, diagnostics, intraocular lenses, cell culture, sutures, and orthopedics) [54–56]. Because of the variety in monomers’ structures, combination, comonomer composition, and molecular weights, different architectures of PSAMs can be formed by immobilizing polymeric units as random or block copolymers onto the surface. Fig. 1.2B illustrates the different conformations of copolymer chains grafted on surfaces through various active sites. In PSAMs, only a small segment of the polymeric construction is attached to the surface, and the rest of the chain is free to control the properties and functionalities of the whole surface. Polymers including poly(ethylene glycol) (PEG), polyurethane (PU), poly(vinyl pyrrolidone) (PVP), polyethylene oxide (PEO), poly(2-methoxyethyl methacrylate) (PMEMA), and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as well as their block copolymers have been constantly used in surface modification [57–62]. Polymers with typical functional groups could be anchored onto solid surfaces by hydrogen or covalent bonding and also through electrostatic interactions. Due to the presence of hydroxy groups on most inorganic oxide surfaces, polymeric units such as poly(2-vinylpyridine) (P2VP), P4VP, and poly(4-hydroxystyrene) (PHOST) can easily form hydrogen bonds or covalent oxide bonds with oxide surfaces. Polymers with alkoxysilyl active groups in their chains or backbones, including poly(3-trimethoxysilylpropyl methacrylate) (PTMSMA) and poly(3-triethoxysilylpropyl isocyanate) (PIC), might be immobilized onto inorganic oxide substrates by covalent bonds through condensation reactions.

In the case of PSAM formations in aqueous solutions, the hydrophobic side chains (long alkyl or aromatic groups) can be attached to the substrate surface through hydrophobic interactions. The creation of PSAMs using cationic and anionic species with the ability to bind to polymers containing amine or carboxylic acids such as polypeptides or polyelectrolytes is applicable through electrostatic interactions. Thiol or sulfide functionalized polymers can bind to metallic substrates and create PSAMs similar to thiolates [57]. The self-assembled block copolymer structures can provide different combinations with different properties just by choosing various constructive monomers (Fig. 1.2C) [60,63–65]. The uniform block copolymer-based SAM can be developed by using the proper concentration of the dipping copolymer solution. Due to stability, biocompatibility, and dictated properties, polymer brushes are good and proper candidates for biomedical applications such as stem cell expansion, implant materials, biosensors, and antifouling medical devices [64,66–70].

Medical devices, including biosensors, heart valves, and catheters, require stable protein-resistant surfaces inside biological fluids [71,72]. In this case, PSAMs made of low fouling hydrophilic segments such as PEG, polyamides, polysaccharides, polybetaines, and polyampholytes resist protein adsorption [73]. Surman and coworkers considered the stability of various PSAMs including poly(carboxybetaine acrylamide, poly[oligo(ethylene glycol)methyl ether methacrylate], poly(2-hydroxyethyl methacrylate) (PHEMA), and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) in exposure to blood plasma. Their study exhibited the highest stability for PHPMA, which can be preserved for 2 years [73].

The surface modification of polymer brushes to provide a selectively binding surface is desirable in some applications, such as biological component detection. These types of surfaces can be modified by chemical methods, including click chemistry or carbodiimide to selectively bind to peptides, proteins, enzymes, or nucleic acids. Cullen et al. created a biosensor based on covalently attached PVDMA to RNase A enzyme [74]. In another study, P(GMA-r-HEMA) brush tethered to the surface of poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) for the covalent sequestration of glucose oxidase was used in cooperation with a catalytic electrode to detect glucose concentrations [75]. The modified polymer brush surfaces can be applied for controlling cell–material interactions. In case of using PSAMs, cell–material interactions are mediated by nonspecific protein adsorption or through modification of a low fouling surface to present cell adhesive peptides [76–83]. Villa-Diaz et al. used PSAMs based on poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) to support human pluripotent stem cells (hPSCs) for several passages [83].

1.4 Self-assembled monolayers based on dendrons

Dendrons or dendrimers are a family of synthetic polymers with regular-branched tree-like structures [84–88]. The branched structures provide a precise surface with controllable properties and an interior structure with the ability to host different materials. The size and molecular weight of dendrons can be controlled by manipulating the repeated branches [89]. The unique features of dendrons make them attractive nanomaterials for different applications including catalysis [90,91], pharmacology [92], biosensors, and biomedicals [93–96]. The combination of dendrons’ chemical structures with self-assembly methods to form SAMs has been studied by several groups. The introduction of various functions to dendrons has affected their practicability regarding their applications. The constructive segment of dendrons consists of several sidechain terminals. The functionalization of these segments, followed by increasing their number on the surface of the dendron molecule, results in the magnification of the functions and their functionalities (Fig. 1.3A).

Figure 1.3 (A) Representation of dendrons’ structural and chemical features (a) and the level of dendronization (b) [97]. (B) Illustration of dendron-based application. Prostate-specific antigens (PSA) is captured by 8A6 antibodies and detected by 5A6 antibodies. (C) Scheme demonstrating dendron modification. (D) Dendron-thiol structures and the observation of the self-assembled monolayer on a gold surface by STM. (B) Reprinted from D. Roy, S.H. Kwon, J.W. Kwak, J.W. Park, Seeing and counting individual antigens captured on a microarrayed spot with force-based atomic force microscopy, Anal. Chem. 82(12) (2010) 5189–5194 © 2010, American Chemical Society. (C) Reprinted with permission from D. Roy, J.W. Kwak, W.J. Maeng, H. Kim, J.W. Park, Dendron-modified polystyrene microtiter plate: surface characterization with picoforce AFM and influence of spacing between immobilized amyloid beta proteins, Langmuir 24(24) (2008) 14296–14305 © 2008, American Chemical Society. (D) Reprinted with permission from L. Zhang, B. Zou, B. Dong, F. Huo, X. Zhang, L. Chi, et al., Self-assembled monolayers of new dendron-thiols: manipulation of the patterned surface and wetting properties, Chem. Commun. (Camb.) (19) (2001) 1906–1907 © 2001, Royal Society of Chemistry.

The immobilization of factionalized dendrons onto the gold surface is one of the most studied areas showing significant enhancement in sensitivity and selectivity of the chemical sensors due to increased density of functional groups [89,93,98–101]. Moreover, the unique features of dendrons make them controllable lateral spacers between functional groups on a surface. For example, gold surface modification with first- and second-generation dendrons could enhance the alpha-helix formation of the immobilized oligopeptides [101], organo-siloxane thin films of SiCl3-terminated dendrons on mica by spin-coating [102], and allyl-terminated dendrons on hydrogen-terminated silicon surfaces [103].

Dendrons-based SAMs possess unique features such as efficient immobilization, facile decoration, high reactivity and selectivity, and low nonspecific bindings to target molecules, which increased their use. For example, dendrons-based SAMs can enhance the performance of DNA microarrays by providing the probe nanosized sites [104,105]. The dendron-modified surfaces could be utilized for developing atomic force microscope results by providing well-defined, homogenous, and controlled spacing surfaces for biomolecule immobilization [106–109]. Dendrons-based SAMs can be used for mRNA distribution mapping at the single-molecular level [110]. The differential expression of mRNA through its distribution within a cell and crucial part in localized protein is being used for cellular and tissue differentiation [111]. The platform with immobilized DNA probe strands on third-generation dendrons with controlled spacing on the surface was used for mapping the distribution of target RNA in a mouse embryonic brain tissue section as a model [110]. The results illustrated simple force curves and narrow histograms for DNA–DNA interactions, which proved the dendron-modified platform’s ability to measure RNA–DNA interactions. Protein detection in very low concentrations is essential for laboratory and clinical studies for protein biomarker detection [112]. For this purpose, force-based AFM containing dendron-modified surfaces was employed to detect captured antigens on a microarray (Fig. 1.3B) [113].

The dendron-modified surfaces have been utilized in polystyrene microtiter plates to reduce the nonspecific binding of biomolecules and provide the lateral spacing between the immobilized biomolecules (Fig. 1.3C) [114]. The obtained results of applying the modified microtiter plates show higher signals on ELISA (10 times higher signals in comparison with other conventional plates) due to covalent immobilization of proteins onto the plates with sufficient lateral spacing.

SAM structures based on dendrons have been used in DNA sensors using electrochemical impedance spectroscopy (EIS) [115]. The system’s measured Rct (charge transfer resistance) exhibited a 2.71 times increase after the hybridization for the dendron-modified electrode. In comparison, it only increased 1.33 times for the SAM electrode in the presence of [Fe(CN)6]³–/⁴– as an electrolyte. The possible reason for such a behavior is related to the difficulty of the probe ion to penetrate the SAM layer that is compacted and hybridized with the target DNA as a probe. Thus, dendron-modified electrodes provide a better platform for studying DNA hybridization with impedance spectroscopy. Moreover, Day et al. reported the formation of polyamidoamine (PAMAM) dendrimers conjugated DNA molecules that can be used to produce SAMs on gold substrate [116]. This platform with high thermal stability was specific to target DNA in solutions due to the presence of flexible dendrimers on the monolayer-coated gold surface.

Zhang et al. reported an SAM structure based on thiol-functionalized dendrons, forming patterned stripes with nanometer-sized features and long-range order. The mentioned SAM platforms could be improved by thermal annealing [117]. In another study by the same group, a stable and unique assembly thiol-functionalized SAM structure was prepared using two types of building blocks (Heptane chains and oligo(ethylene oxide)) with different hydrophobicity with locally controlled hydrophobicity [117]. Carrying hydrophobic and hydrophilic groups by the immobilized SAMs of dendron-thiols on a gold surface could form pore structures with self-organizing ability in smooth areas. This precisely tailored structure with local controlled hydrophobic and hydrophilic moieties could be used in various fields such as bioapplications.

1.5 Self-assembled monolayers based on lipids

Self-assembled lipid layers are naturally essential structures with important biological functions, including cell–cell and cell–extracellular matrix interactions, signal transduction, receptor binding, and molecular transport, in all living organisms [118]. Self-assembly mechanism and their incorporation into novel biomimetic structures were intensively studied by researchers due to their biocompatibility, biodegradability and stability properties [119–121]. Self-assembly of lipids spontaneously occurs to form larger supramolecular structures such as cell membranes. Hydrophobic interactions are primarily responsible for the structure of these biological networks. The other driving forces for forming self-assembly structures by various lipids such as triacylglycerols and phosphoacylglycerols are hydrogen bonding, van der Waals forces, electrostatic interactions, π–π stacking, and host–guest interactions. Biologically inspired self-assembled structures by lipid building blocks have been largely developed for many technologies, including diagnostics, therapeutic nanocarriers, and biosensors [122,123]. Self-organization of lipids provides well-identified membrane systems, including monolayers, bilayers, and