Thesis
Reference
Adhesion and mechanics of dendronized polymers at single-molecule level
GREBIKOVA, Lucie
Abstract
In this thesis, the physical properties of dendronized polymers (DPs) were investigated by means of surface sensitive techniques, namely atomic force microscopy (AFM) and reflectometry. The adhesion and mechanics of DPs were studied at the single-molecule level by AFM. A novel nano-handling technique based on AFM is presented. This technique was used to study the force response of DPs of different generations. It relies on combination of AFM imaging and force measurements. AFM was further employed to investigate the persistence length of DPs and combined with optical reflectometry to study the adsorption of DPs on planar surfaces.
GREBIKOVA, Lucie. Adhesion and mechanics of dendronized polymers at single-molecule level. Thèse de doctorat : Univ. Genève, 2015, no. Sc. 4802
URN : urn:nbn:ch:unige-743694
DOI : 10.13097/archive-ouverte/unige:74369
Available at:
http://archive-ouverte.unige.ch/unige:74369
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE FACULTÉ DE SCIENCES Section de chimie et biochimie Professeur Michal Borkovec Département de chimie minérale
et analytique
_____________________________________________________________________________
Adhesion and Mechanics of Dendronized Polymers at Single-Molecule Level
THÈSE
présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès science, mention chimie
par Lucie Grebíková
de
Brno (République Tchèque)
Thèse N° 4802
GENÈVE Atelier ReproMail
2015
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Content
1 Introduction ... 9
1.1 Polymer adsorption ... 9
1.2 Single molecule mechanics of polymers ... 13
1.3 Dendronized polymers ... 17
1.4 Surface sensitive techniques ... 19
1.4.1 Atomic force microscopy ... 19
1.4.2 Reflectometry ... 23
1.5 Outline of the thesis ... 26
2 Interactions between Individual Charged Dendronized Polymers and Surfaces ... 28
2.1 Introduction ... 28
2.2 Experimental section ... 29
2.3 Results and discussion ... 32
2.4 Conclusions ... 43
3 Single-Molecule Force Measurements by Nano-Handling of Individual Dendronized Polymers... 44
3.1 Introduction ... 44
3.2 Experimental section ... 45
3.3 Results and discussion ... 47
3.4 Conclusions ... 57
4 Recording Stretching Response of Single Dendronized Polymer Chains Adsorbed on Solid Supports ... 59
4.1 Introduction ... 59
4.2 Experimental section ... 62
4.3 Results and discussion ... 65
4.4 Conclusions ... 79
5 Persistence Length of Charged Dendronized Polymers ... 81
5.1 Introduction ... 81
5.2 Experimental section ... 82
4
5.3 Results and discussion ... 84
5.4 Conclusions ... 94
6 Adsorption of Dendronized Polymers on Planar Water-Silica Interface Investigated by Combining Optical Reflectometry and Atomic Force Microscopy ... 95
6.1 Introduction ... 95
6.2 Experimental section ... 96
6.3 Results and discussion ... 99
6.4 Conclusions ... 108
7 Conclusions ... 109
8 Acknowledgements ... 110
9 References ... 111
5
Abstract
In this thesis, the physical properties of dendronized polymers (DPs) were investigated by means of surface sensitive techniques, namely atomic force microscopy (AFM) and reflectometry. The adhesion and mechanics of DPs were studied at the single-molecule level by AFM. This technique was further employed to investigate the persistence length of DPs and combined with optical reflectometry to study the adsorption of DPs on planar surfaces. The first chapter describes the theoretical background of the polymer adsorption, polymer mechanics at the molecular level and the surface sensitive techniques used for the investigation of DPs.
The second chapter shows the influence of the generation of the DP, surface hydrophobicity and ionic strength on the adhesion of DPs. The adhesion increased with increasing DP generation, surface hydrophobicity and ionic strength. The generation effect can be explained by increased polymer-surface contact area which leads to stronger adhesion. The surface hydrophobicity and ionic strength investigation suggests that the interaction between DPs and surfaces is predominantly driven by non-electrostatic interactions.
In the third chapter, a novel nano-handling technique based on AFM is presented. This technique was used to study the force response of DPs of different generations. It relies on combination of AFM imaging and force measurements. A polymer chain is adsorbed on a properly functionalized substrate, imaged by AFM, and subsequently picked up by the AFM tip at one of the ends. The single polymer chain can be then stretched many times, providing a large number of force curves. The mechanical response of DPs could be accurately described by the freely jointed chain model, demonstrating that the elastic response of DPs changes with the polymer generation and ionic strength.
The fourth chapter extends the investigation of the mechanical response of DPs. The nano-handling technique is compared with the conventionally used pulling methods, namely classical pulling and fishing techniques. As demonstrated in the third chapter, the nano-handling technique ascertains that the force experiments are truly carried out at the single-molecule level. The other methods do not provide such a direct way to verify if the force response originates from a single polymer molecule. This chapter shows that the conventional methods not always give a single molecule stretching response. The response was found to be substrate and solution conditions dependent.
The fifth chapter focuses on the identification of the persistence length of DPs by AFM imaging. The persistence length of DPs adsorbed on various surfaces was obtained in electrolyte solution of different ionic strengths using the decay of the bond-bond correlation function and the internal end-to-end
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distances. The persistence length of DPs increases with increasing generation and decreasing ionic strength. These findings apply also to different surfaces. However, it was found that the specific polymer- surface interactions strongly influence the magnitude of the persistence length.
The sixth chapter reports on the adsorption of DPs on planar silica substrates. The effects of polymer generation and ionic strength on the adsorption were investigated by combining optical reflectometry and AFM. With increasing ionic strength and generation, the adsorbed amount significantly increases. This finding can be rationalized by an increase in the screening of electrostatic repulsion between the adsorbed polymer chains. AFM imaging supports well the results obtained from reflectrometry, showing significantly denser polymer layers with increasing both ionic strength and DP generation. Furthermore, AFM image analysis showed that the conformation of adsorbed polymer chains is affected by the surface coverage.
In the seventh chapter, the general conclusions of the thesis are discussed.
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Résumé
Dans cette thèse, les propriétés physiques de polymères dendronisés (PDs) ont été étudiées au moyen de techniques de caractérisation de surface, c'est-à-dire la microscopie à force atomique (MFA) et la réflectométrie. L’adhésion et les propriétés mécaniques des PDs ont été étudiées à l’échelle moléculaire par MFA. Cette technique a été utilisée en particulier pour identifier la longueur de persistance de PD en combinaison avec la réflectométrie pour étudier l’adsorption des PDs sur des surfaces planes. Le premier chapitre décrit la base théorique de l’adsorption des polymères, leurs propriétés mécaniques à l’échelle de molécule unique et les techniques de caractérisation de surface utilisées dans le cadre de ces études.
Le deuxième chapitre montre l’influence de la génération du PD, de l’hydrophobicité de la surface et la force ionique sur l’adhésion des PDs. L’adhésion est augmentée avec la génération, l’hydrophobicité de surface et la force ionique. L’effet de la génération peut être expliqué par l’augmentation de la surface de contact entre le polymère et la surface, ce qui induit une adhésion plus importante. L’étude de l’effet de l’
hydrophobicité de la surface et de la force ionique met en évidence que l’interaction entre les PDs et la surface est principalement contrôlée par des interactions non-électrostatiques.
Dans le troisième chapitre, une nouvelle technique de nano-manipulation basée sur la MFA est présentée. Cette technique permet la mise en évidence des propriétés mécaniques des PDs de générations différentes. Elles ont été identifiées en combinant la MFA avec des mesures de force. Une chaîne de polymère est adsorbée sur une surface convenablement fonctionnalisée. Une image est réalisée par MFA, puis le polymère est détaché par l’une de ses extrémités au moyen de la pointe MFA. Le polymère peut être manipulé plusieurs fois de cette façon, ce qui permet d’obtenir un nombre significatif de courbes de force. La réponse mécanique des PDs a donc pu être décrite avec précision par le model de pelote aéatoire. La réponse élastique des PDs dépend donc de la génération du DP et la force ionique.
Le quatrième chapitre étend l’étude de la réponse mécanique des PDs. Les résultats obtenus par nano- manipulation sont comparés à ceux obtenus par les techniques classiques pour manipuler les polymères, c'est-à-dire la technique d’élongation classique et la technique de pêche. Comme démontré dans le troisième chapitre, la technique de nano-manipulation permet d’avérer que les mesures de force sont vraiment effectuées à l’échelle de molécule unique. Les autres méthodes ne permettent pas d’obtenir directement un tel résultat afin de vérifier que la réponse est celle d’une molécule individuelle. Il a de plus été établi que, pour cette methodes, la réponse dépend des propriétés de la surface et de celles de la solution.
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Le cinquième chapitre se focalise sur l’étude de la longueur de persistance des PDs par l’imagerie MFA. La longueur de persistance des PDs adsorbés sur des surfaces diverses a été mesurés dans des électrolytes de force ionique différente en utilisant la fonction de corrélation qui décrit les liaisons et les distances bout-au-bout intramoléculaires. La longueur de persistance des PDs augmente avec l’augmentation de la génération et une diminution de la force ionique. Ces conclusions s’appliquent aussi à l’interaction avec des surfaces aux propriétés diverses. De plus, il est démontré que les interactions spécifiques entre le polymère et la surface influencent fortement la longueur de persistance.
Le sixième chapitre relate l’adsorption des PDs sur des surfaces planes de silice. L’effet de la génération du polymère et la force ionique a été étudié en combinant la réflectométrie et la MFA. La quantité de polymère adsorbée augmente significativement avec l’augmentation de la force ionique et la génération du PD. Cette conclusion peut être rationalisée par l’augmentation de la répulsion électrostatique entre les chaînes de polymères adsorbés. Les résultats d’imagerie MFA sont en accord avec ceux obtenus par réflectométrie, montrant que la densité de surface augmente avec la force ionique et la génération. De plus, l’analyse des images de MFA a montré que la conformation des chaînes de polymères adsorbés est affectée par le recouvrement en surface.
Dans le septième chapitre, les conclusions générales de la thèse sont discutées.
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1 Introduction
1.1 Polymer adsorption
Understanding of polymer adsorption onto surfaces forms the scientific basis of many biological and technological applications. The ability to describe and control polymer adsorption at various substrates is crucial to establish molecular design principles of antifouling surfaces [1-5], fabricate biosensors [6, 7], prepare low-friction low-wear surfaces and coatings [8], polymer-based technological glues [9, 10], and develop innovative adhesives for biomedical applications [9].
Polymer adsorption is a complex phenomenon, which touches upon both theoretical and experimental areas of research. Numerous studies on polymer adsorption have been published and are directed at determining the influence of polymer and surface charge densities, polymer conformation at the surface, polymer architecture, surface characteristics, and the interaction force of adsorbed polymers at surfaces [4,
11-15]
. With the current developments in atomic force microscopy (AFM) techniques, it has now become possible to tackle polymer adhesion at the true molecular level and open up the possibility to systematically investigate polymer adsorption properties and different intermolecular interactions. For this purpose, single molecule force spectroscopy (SMFS) based on AFM has been developed [14, 16-19]. In SMFS, a polymer chain is firmly attached to the tip of an AFM cantilever and subsequently stretched between the AFM tip and an opposing surface. The force exerted on the cantilever is measured as a function of the distance from the surface as the polymer is pulled perpendicularly to the surface (see Figure 1.1). Force-extension curves then reflect the desorption of a polymer chain from the surface. The principle of the AFM measurement is explained in greater detail in the Chapter 1.4.1.
In desorption experiments, the shape of recorded force curve strongly depends on the dynamics of the system, ranging from saw-tooth pattern to long plateaus [20]. The polymer chains are linked to the substrate either covalently or by non-covalent interactions. In both cases, the rupture of binding sites with the surface leads to extension of the polymer chain by the length of the strand between one and the next binding sites. Depending on the internal dynamics of the probed bonds and the applied force loading rates, the adhesive force acting between the polymer chain and the substrate can be obtained from the force-extension profile as the height of individual unbinding peaks, or from the height of the desorption plateaus (Figure 1.1).This is supported by theory, suggesting that the shape of the force-extension profile for continuous desorption of adsorbed polymer chains from a solid substrate (i.e. unbinding of multiple bonds in series) depends on the force loading rate [21].
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Figure 1.1 Schematic representations of desorption of a polymer chain from a solid substrate. The polymer is coupled to the surface through numerous interaction sites (bonds). Depending on the loading rate and the internal dynamics of the probed bonds, the force-extension profile shows a saw-tooth pattern with individual peaks of distance s corresponding to individual bond rupture (a), or a desorption plateau of constant force with the length L of the desorbing polymer chain (b).
Bond rupture forces are generally nonequilibrium values, depending on the intrinsic lifetime of the bond, the temperature, and on the measurement time [22, 23]. At pulling rates much faster than internal dynamics of the probed bones, each detachment of polymer-surface interaction site provides a peak of a saw-tooth pattern (Figure 1.1a). The rupturing of polymer-surface bonds is an irreversible process and happens under nonequilibrium conditions. These peaks are typically observed for covalent bonds [22], specific receptor-ligand systems [24, 25], as well as for the rupture of protein domains [26, 27]. In case that the distance between the different binding sites on the polymer is decreasing, it becomes more difficult to resolve the single rupture events and the force profile has a plateau-like shape. The measurement still takes place in nonequilibrium, the rupturing of bonds is irreversible and loading rate dependent process [22].
For lower pulling rates, the individual bonds dissociate and reassociate on much faster time scale and the heights of individual peaks related to the consecutive unbinding events decrease. In some cases, one can then observe a desorption plateau of constant force corresponding to the equilibrium desorption of the polymer from the surface segment by segment (Figure 1.1b). The desorption of the polymer is then a reversible process and the measured force is a measure of the polymer-surface adhesion free energy.
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Continuous desorption plateaus are observed when the polymer is only weakly adsorbed, e.g.
polyelectrolytes bound to charged surfaces [12, 14, 28].
During the desorption experiments, plateaus of constant force with several steps can be observed in the force-extension curve [29]. Multiple molecules of different lengths are simultaneously adsorbed on the surface and the different steps represent the desorption of individual molecules one after the other. Each time, a step in the desorption force is recorded when one molecule is completely desorbed, until the moment when the last polymer is fully detached. This means that in the case of multiple events, the last step reflects the detachment of a single polymer from a substrate. The plateau length corresponds to the length of the adsorbed molecule, whereas the height of the plateau to the desorption force that is necessary to desorb one or several polymer chains from the surface.
Not only the dynamics of the system, but also the interaction between the polymer and a solid substrate can affect the shape of the force-extension profile. Based on the polymer-surface interaction, the conformation of an adsorbed polymer chain can go from a train-like structure to a polymer brush. In the intermediate region, a polymer chain can form a loop or tail structure (Figure 1.2) [30]. For some polymer systems, Zhang et al. [20] related the pattern of the force-extension profiles to adsorption conformation of polymer chains on the interface. A force profile showing a single peak corresponded to the detachment of a tail structure, a saw-tooth pattern corresponded to the detaching of several loop structures in series, and a plateau of constant force came from the detachment of a train-like structure. Nevertheless, the important factors determining the adhesion behavior of polymers, and subsequently force-extension profiles, are the nature of the polymer-surface bond and the solution composition [20, 23].
Figure 1.2 Schematic representation of adsorption conformations of a polymer chain.
The nanostructure of materials and surface modifications play an important role in interaction of polymers with solid substrates. The control of surface properties allows one to tune molecular adhesion properties. AFM-based single molecule force spectroscopy has been used to systematically investigate the adsorption process of individual polymers on generic surfaces. Different types of interactions (e.g., electrostatic or hydrophobic interactions) influence the adhesion process on a variety of surfaces [17]. For example, the polymer desorption was studied on self-assembled monolayers (SAMs) with different
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terminal groups [4, 31]. SAMs represent easily accessible systems which allow one to investigate the adsorption-desorption process on well-defined and controlled surfaces. Geisler et al. [12, 16] studied single- polymer adhesion in terms of their adsorption strength on various materials, including the effects of surface composition, hydrophobicity, electrical properties and surface roughness. From a macroscopic point of view, by increasing the surface roughness the effective surface area increases which is supposed to enhance adhesion. Surprisingly, the experiments performed with a molecule on either smooth or rough surfaces show that the surface roughness has no effect on the adhesion of a single polymer [12]. Most material properties such as conductivity, surface potential, and composition hardly influence single- molecule adhesion under equilibrium conditions [16]. Solvent-related properties have greater influence on the adhesion strength but still lead to less than 50% difference [15, 16]. These studies have shown that surface modifications enable the fine-tuning of the surface adhesion of polymers but as long as the polymers are still weakly attached to the substrate, these effects are only marginal [12, 15-17, 32]
. The same conclusion applies also for the effect of molecular architecture on single polymer adhesion [13]. A significant increase in adhesion can be observed when many polymers interact not only with the surface but also with each other via intra- and inter-chain entanglements, for example hydrogels, polymer films and brushes. Adsorption properties of these materials can be also changed by external stimuli [7, 33, 34].
Traditional single molecule approach to investigate the polymer adsorption-desorption process relies on a polymer chain firmly attached to the tip of an AFM cantilever. The force exerted on the cantilever is measured as a function of the distance from the surface as the polymer is pulled perpendicularly to the surface [14, 19, 27, 28, 35-37]. With advances in AFM technology, it has become possible to control the tip position and direction with high precision. This has led to experiments in which the adsorbed polymer can be pulled at different positions along the backbone and also pulled from the surface along different directions [8, 38-40]. For example, a single polymer chain was pulled laterally over the substrate in order to enhance polymer adsorption [38] or to study single polymer friction [8, 39]. There are theoretical and computational studies [41, 42] focusing on the effect of the angle at which the force is applied, ranging from perpendicular to parallel to the surface. These models predict that depending on the angle, the polymer favors either adsorption or desorption. There is a critical angle value beyond which the polymer cannot be desorbed by applying a force. Serr and Netz [38] have shown that pulling the polymer parallel to the surface enhances adsorption, pulling the polymer perpendicular by strengthening the force increases the tendency to desorb.
13 1.2 Single molecule mechanics of polymers
Single polymers show a strongly nonlinear behavior when they are stretched by an external force.
Stretching a polymer molecule causes two kinds of restoring forces. The low force regime (short extensions) is dominated by purely entropic contributions. If a single polymer chain adopts a random coil conformation in solution, the Brownian molecular motion causes a permanent fluctuation of the molecule around a mean equilibrium conformation. Extension of the molecule reduces the number of possible conformations, which causes a loss of conformational entropy. The high force regime is dominated by enthalpic contribution as large chain elongations lead to a tension of the molecular backbone. Bonds become stretched and deformed in the direction of the pulling force, and the corresponding enthalpic elasticity is recorded in addition to the entropic forces. In the medium range, the rupture of salt bridges and intramolecular hydrogen bonds may occur, eventually leading to conformational changes of the polymer. The mean values describing the conformation of a polymer chain in solution can be derived by statistical mechanics [43, 44]. Two models are commonly used for describing the elasticity of a polymer chain: the freely jointed chain model (FJC) [44] and the worm-like chain model (WLC, or Kratky-Porod model) [43, 45].
In the FJC model, a polymer chain is divided into n rigid Kuhn segments of equal length , connected through flexible joints without any long range interactions (Figure 1.3). The segments are freely jointed without restrictions for their spatial arrangement. Each segment is independent of every other, including the nearest neighboring bonds, and can be oriented in every direction with equal probability. The directions of neighboring bonds are thus completely uncorrelated:
i j 0, i j
l l (1.1)
where liand ljrepresent the bond vectors.
The contour length of the polymer chain is given byLn. The partition function Zof a freely-jointed polymer chain subjected to an external forceF, as a function of the extensionx, can be written as:
lnZF x k T R
(1.2)
where k is the Boltzmann constant, and T the absolute temperature. In most cases, it is not possible to derive an analytical expression forZ, therefore an additional term, stretching energyF x , is introduced into chain’s partition function:
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1
, , /
n
E F x kT
l l
Z
e l1ln dl1dln (1.3)Where l1, , lnare the bond vectors representing the n segments of length. E
l1, , ln
is the energy of a given conformation which is a constant as there is no interaction between individual segments in FJC model. The extension xcan be then calculated as a function of the external forceFfrom:lnZ x k T
F
(1.4)
Based on the Eq. (1.3) and Eq. (1.4), the extension of a polymer chainx can be expressed as a function of the applied force Fby:
x coth F k T
L k T F
(1.5)
This model solely accounts for the entropic elasticity of the chain’s backbone. Since elastic deformations are neglected, the polymer chain cannot be stretched by more than the contour length. This equation is then a good approximation only for small extensions (xL). For large extensions, the deformation of bonds and bond angles will result in an effective increase in the segment length.
Therefore, the enthalpic contribution to the restoring force of the polymer chain needs to be taken into account and the extended FJC model has been proposed [46].
Figure 1.3 Schematic representations of (a) freely jointed chain (FJC) model composed of n rigid segments (with the Kuhn length ) coupled by flexible joints and (b) extended FJC model where the parts of the chain are replaced by elastic springs to include enthalpic effects.
The extended FJC model considers the polymer molecule as n identical elastic springs in series and introduces an additional parameterK, the elasticity constant, to describe each segment [Eq. (1.6)].
x coth F k T F
L k T F K
(1.6)
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The extended FJC model has been successfully used to describe the stretching behavior of various synthetic polymers, such as poly(acrylic acid) [47], poly(ethylene-glycol) [35], poly(vinyl alcohol) [48], polyacrylamide [49], poly(ferrocenyldimethylsilane) [50], dendronized polymers [51], as well as many polysaccharides, including cellulose [52], carboxymethylcellulose [53] and dextran [54].
The WLC model neglects any discrete structure along the chain and describes a polymer as a homogeneous, continuous string r
s of constant bending elasticity B (Figure 1.4) [43, 55]. The elastic bending energy EBis proportional to the square of the curvature and is given by:2 2
2 2 B
E B ds
s
r (1.7)where s
ris the unit tangent vector and sis the coordinate measured along the polymer contour.
The molecule is constantly in motion, bending in random directions. The interplay between Brownian motion and rigidity is determined by the persistence length Prepresenting the flexibility of the polymer chain.
Figure 1.4 Schematic representations of the worm-like chain (WLC) model. (a) A polymer chain is described as a
continuous stringr s , with s taking values from s0to sL. The chain direction is preserved on the length scale of the persistence length P. (b) The directional correlation between two segments in a polymer separated by the distance s is given by Eq. (1.9).
The persistence length is defined as the decay length of the directional correlation along the polymer chain and is given by:
P
B
k T
(1.8)
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It is a direct measure of the average local conformation of a polymer chain and below this length the polymer can be considered to be linear. If sis reasonably large, the chain corresponds to a Gaussian chain and the average correlation of the tangent vectors decreases exponentially withs:
P
cos 0 exp s
s s
r r (1.9)
Where r
s and r
0 are the unit vectors tangent to the chain at positions sand 0.
s is the angle between the tangents along the chain separated by the distances. WhensP , Eq. (1.9) leads to cos
s 1, the angle
s fluctuates around zero and the chain segments have nearly same direction. In the case ofP s, Eq. (1.9) results in cos
s 0, indicating that
s can be anything from 0° to 360° with equal probability, i.e. the memory of the chain direction is lost once Pis exceeded.For small extensions, and in the limit of flexible chains (LP), the persistence and Kuhn length are related by 2P[43, 55].
The exact force-extension relation of a wormlike chain can only be determined numerically, but a commonly used analytical approximation is given by the formula of Marko and Siggia [56, 57]:
2
P
1 1
4 1 4
k T x x
F L L
(1.10)
Although entropic and enthalpic contributions are combined in this approach, the extension is limited by the contour length of the polymer [58]. The model does not hold in the high force regime where the segment length increases due to the stretching of the covalent bonds. In analogy to the FJC model, an additional stretching term Kis introduced. The parameter K is appropriate for intrinsic enthalpic contributions and denotes the specific stiffness of the polymer chain. The extended WLC models established by Odijk and Wang offer a good applicability over a wide range of applied forces and can be found elsewhere [59, 60].
The WLC and extended WLC models have been effectively used to describe the force-extension behavior of various polymers, such as DNA [60-62], proteins [62, 63], poly(methacrylic acid) [64], and poly(vinyl alcohol) [65].
In the presence of an external force, polymers often undergo conformational and configurational transitions upon stretching. This is marked by a deviation from the FJC and WLC model in the mid-force regime. The low force regime (below the conformational transition) and the high force regime (above the
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conformational transition) can be fitted separately according to the FJC or WLC model. Typical examples are polysaccharides where high stretching forces bring about conformational transitions in the pyran ring
[52, 54, 66]. An external force can lead to a repeated unfolding of protein domains as reported for titin and tenascin [26, 54]. Furthermore, stretching can induce considerable changes in the secondary structure of single polymer chains. Experiments on poly(ethylene glycol) [35] in water indicated the deformation of the helically folded equilibrium conformation of the polymer due to overstretching the hydrogen-bonded solvation superstructure. Similar transitions were also observed for xantan [67] and PVP [68]. More complex system is double-stranded DNA showing a highly cooperative conformational transition from its natural form (B-DNA) to an overstretched conformation (S-DNA), upon which the length of the molecule is approximately doubled [46].
Changes in the mechanical response of polymers can be also induced by the changes in the environmental conditions, i.e. external stimuli. The stimuli can be chemical (pH, ionic strength, solvent) or physical (temperature, electric or magnetic field, mechanical stress) [28, 47, 50, 61, 69-72].
1.3 Dendronized polymers
Dendronized polymers (DPs) represent nanostructured molecular objects with high level of structural complexity at the interface between materials and biosciences. In recent years, DPs have attracted considerable interest for their potential application in biology and material sciences, including the development of optical devices [73, 74], biosensors [75], supports for enzymes or nucleic acids [76, 77], and drug delivery systems [77, 78]. They comprise linear backbones carrying repeatedly branched dendrons of varying generation as the side chains. Free ends make up more than 50% of the monomers in a dendronized polymer, with the spatial distribution favoring the exterior envelope of the chain. A large number of end groups permits then the control of solubility as well as further chemical modifications [79]. There are two principally different synthetic approaches to dendronized polymers, i.e. “attach-to”
route and macromonomer route [79, 80] (Figure 1.5). In the attach-to route, two types of trifunctional building blocks, polymerization (P) and dendronization (D) units, are used. P units with two blocked functionalities are polymerized thus forming a first generation of DP PG1 (Figure 1.5a and b). The resulting polymer is deblocked (Schema 1c) and reacted with D units to create a second generation PG2 (Figure 1.5d). Higher generations are created by repeating the de-protection step followed by reaction with blocked D units. In the macromonomer route, the P units already carry a dendron of generation in question (Figure 1.5e) and their polymerization leads directly to this generation of DP (Figure 1.5f). The stepwise addition, “attach-to” route, allows the production of long high generation DP whereas the
18
polymerization of dendronized monomers, the macromonomer route, only creates short DPs of higher generation, due to the bulkiness of the P units.
Figure 1.5 The two synthetic routes to dendronized polymers, “attach-to” route and macromonomer route [80]. The homologous series of different generations of DPs relies on the same chemistry but differs in the number of monomers in the side chain [81]. Therefore, DPs allow systematic, generation-dependent study of the variation in thickness, persistence length and other physicochemical properties. The thickness of DPs allows distinguishing between species of different generation using atomic force microscopy when coadsorbed onto a single substrate. DPs adsorb as weakly deformed cylinders, the degree of deformation varying with generation. Compared to dendrimers and bottle brushes of linear chains, the cylindrical geometry of DPs provides smaller volume to a side chain, thus leading to stronger chain stretching and weaker deformability. The highly crowded DPs are close to their maximum extension, thus the higher the generation is, less deformable coronas and higher backbone rigidity is expected [81]. Numerous studies on DPs have addressed their responsive behavior [51, 82-84], their conformation [85], the dimensions of adsorbed chains [81], self-folding of single charged polymer chain [86], and their interaction with surfaces [37].
19 1.4 Surface sensitive techniques
1.4.1 Atomic force microscopy
The atomic force microscopy (AFM) is the principal technique used in this study to investigate the conformation of DPs on solid substrates, their interaction with surfaces and mechanical response of individual polymer chains at the single-molecule level.
Since the invention of AFM in the mid 1980s by Binnig et al. [87], the technique has developed into a powerful and versatile tool intensely used for the study of the surface topography at atomic and nanometer scale, the chemical structure of a molecule [88], the high resolution imaging of DNA, proteins and polymers [89-91], the inter- and intramolecular interactions in surface-immobilized systems [27, 54, 92-94], the mechanical properties of polymers [28, 95-97], and more. In this thesis, AFM was employed to measure forces and surface topography; both approaches are discussed in the following.
Figure 1.6 (a) Schematic drawing of a force microscope and (b) illustration of a deflection-displacement (piezo position) experiment.
Typically AFM is carried out under ambient conditions, but a great advantage of AFM is the possibility to image the sample in almost any environment, ranging from vacuum, trough gas, to liquid.
AFM probes the surface of a sample with a sharp tip which is located at the free end of a cantilever spring. The cantilever acts as a sensor for the interaction between the tip and a sample (Figure 1.6a). The sample is mounted on a piezoelectric device, which allows the sample to be moved in the vertical direction (z-direction) and scanning the surface in the x-y direction. While the tip scans over the sample,
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the forces between the tip and the sample surface cause the cantilever to bend or deflect. The deflection of the cantilever is detected optically by the deflection of a laser beam focused at the back of the cantilever and reflected to a position-sensitive photodiode.
AFM force measurements. In a force measurement, the x-y position is fixed, while the sample (or tip) is moved up and down by applying a voltage to the piezoelectric device. The cantilever deflection and displacement of the piezo are recorded (Figure 1.6b). During an experiment, the tip is initially far away from the surface, forces are absent and the cantilever is not deflected (Figure 1.6b, Position 1).
Subsequently, attractive surface forces become apparent as the tip approaches the surface and eventually jumps into a close repulsive contact with the surface (Figure 1.6b, Position 2). While the tip and the sample are in contact, the pressure that the tip exerts on the surface increases with the sample displacement. In this region (Figure 1.6b, Position 2-3), known as the constant compliance region, the signal of the photodiode (voltage) and sample displacement are proportional, and the voltage can be directly converted into the cantilever deflection. Upon retraction of the tip and the substrate, the repulsive forces decrease continuously followed by the complete separation of the tip from the surface (Figure 1.6b, Position 4).
These deflection-piezopath data have to be transformed into force-distance curves. The measured piezo position (z0) can be converted into the real distance (z) between the AFM tip and the surface according to Eq. (1.11):
1
zz0s D (1.11)
Here, D is the measured cantilever deflection and s is the slope [voltage/length] of the linear part of the curve reflecting the bending of the cantilever upon contacting and indenting the substrate surface. The force F is then obtained by applying Hooke’s law [Eq. (1.12)] [98]:
0
F kc z z (1.12)
where kc is the spring constant of the cantilever. The minus sign in the equation transforms the negative deflection into a positive force signal. Finally, the force acting on the cantilever is plotted against cantilever-surface distance (z), giving the true force-distance curve.
The cantilever spring constant kc is normally determined from the thermal oscillation spectrum of the cantilever [99, 100], but many other methods [101], such as vibrational based on the geometry of the cantilever
[102], method measuring the resonance frequency of the cantilever before and after adding end masses [103], can be used.
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In the work reported in this thesis, the spring constant was obtained by thermal method in air [99]. The cantilever is positioned far away from the surface, is not affected by long range forces and only vibrates around its equilibrium position due to the thermal fluctuations. If a system is in thermal equilibrium, the ground oscillation of the cantilever has a mean energy equal to 1
2k T (kis the Boltzmann constant, Tis the absolute temperature). Due to thermal motion, the cantilever oscillates with an amplitudex. If the cantilever is modeled as a harmonic oscillator, its resonance frequency is:
0 kc
m (1.13)
where 0is the resonance frequency, kcthe spring constant, and mthe effective mass of the cantilever.
Considering just one degree of freedom for the cantilever (it can move only up and down) and the equipartition theorem:
2 2 2
0
1 1 1
2m x 2kc x 2k T (1.14)
where x2 is the mean square of the thermal cantilever fluctuations. The spring constant can be subsequently obtained as Eq. (1.15):
2 c
k k T
x (1.15)
To exactly determine x2 , it requires integration over the ground oscillation in the power spectrum of the cantilever measured over all frequencies [104].
AFM topography. AFM modes are generally classified as static or dynamic modes, based on the oscillation of the tip during the imaging [105].
In the static mode, the tip does not oscillate and the topography of the surface is generated from the cantilever deflection. There are two basic ways of operation, constant height and constant force. In the constant height mode, the cantilever deflection is detected without a feedback control as the height of the scanner is fixed as it scans and it is used directly to obtain the topographic data. This mode is applicable to very smooth, atomically flat surfaces where the variations in the cantilever deflections (i.e., in applied force) are small. In the constant force mode, the cantilever deflection is kept constant by moving the
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scanner up and down in z direction, generating the image from the scanner motion. Constant force mode is generally preferred as the total applied force to the sample is constant and well controlled.
In the dynamic modes of AFM, the system vibrates the cantilever at or near its free resonance frequency. According to the parameter used to establish the feedback mechanism, two major dynamic modes of AFM are amplitude modulation and frequency modulation AFM [105, 106]. In amplitude modulation AFM, the oscillation amplitude changes as the tip approaches the sample surface and is used as a feedback parameter to obtain the surface topography. On the other hand, in frequency modulation, the cantilever is kept oscillating with a fixed amplitude and the feedback parameter is a frequency shift between the resonance frequency far from the surface and the resonance frequency closer to the surface.
The resonance frequency depends on the forces acting between tip and sample surface. The dynamic (oscillating) AFM modes became widely popular, taking advantage of the signal-to-noise benefits associated with modulated signals. The imaging can be carried out with a small probe-sample force, thus preserving both the sample and the AFM tip.
Figure 1.7 Schematic illustration of the amplitude change as a function of the tip-surface separation.
Throughout this thesis, the amplitude modulation (AC) mode of AFM was used to image the topography of the sample. The cantilever is oscillated (with free oscillation amplitudeA0) typically at or near its resonance frequency with an additional piezoelectric element. When the oscillating cantilever approaches the surface, the forces between the tip and the surfaces cause changes in the oscillation, a damping in the cantilever oscillation (Figure 1.7). The damping leads to a decrease in the resonance frequency and in turn in the amplitude of the oscillation. The feedback loop maintains the amplitude constant. This process involves comparison between the instantaneous value of the amplitude Aiwith respect to a reference value, the set point amplitudeA. An error signal is generated. The goal is to keep
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this error signal as small as possible. Based on the error signal, an integral differential system moves the piezo scanner in zdirection in order to minimize the difference between A and Ai.
Depending on the oscillation amplitude used, AFM can be operated in different regimes, i.e. non- contact, and intermittent-contact regime (Figure 1.8). The interatomic force between the tip and the surface is either repulsive (contact mode) or attractive (non-contact mode). Using a small oscillation amplitude, the cantilever can be held in the attractive regime only. On the other hand, if a large oscillation amplitude is applied, the tip can move from ‘zero-force’ regime, through the attractive regime where is no tip-sample interaction, to the repulsive regime in each oscillation cycle. This technique is known as intermittent-contact AFM (IC-AFM). The cantilever tip, which vibrates at or near its resonance frequency, is brought closer to the sample surface and at the bottom of its travel it just barely hits the sample. IC-AFM has become an important technique as with its introduction it became possible to image soft structures such as polymers, since it is less likely to damage the sample than contact AFM by eliminating lateral forces and is more effective than non-contact AFM by overcoming its fundamental instability in air.
Figure 1.8 Different operating regimes for oscillating AFM modes. Force versus tip-to-sample separation curve illustrating the attractive and repulsive regimes.
1.4.2 Reflectometry
Reflectometry is a simplified form of ellipsometry, which is cheaper, with rather simple setup and uses surfaces easy to prepare and to further functionalize. It enables continuous and quantitative measurements of the polymer or particle adsorption onto flat solid substrates [107-110]. A continuous
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measurement is obtained over short time scales, therefore reflectometry is highly suitable for the measurement of the adsorption kinetics on the time scale of seconds to minutes [110, 111]. With the developments in the reflectometry setup, the technique has even proven to be efficient for probing the ions at a water-silica interface during the formation of the electrical double layer [112], and in biomolecular sensing [113, 114]. A schematic representation of a reflectometry setup is shown in the Figure 1.9.
Figure 1.9 Schematic representation of reflectometry setup.
In this technique, a linearly polarized laser beam is reflected on a surface through an optical prism at a fixed angle of incidence. The beam reflected off the surface passes through a beam splitter (Wollaston prism), and is split into its parallel (p) and perpendicular (s) components with respect to the plane of reflection. Two separate photodiodes continuously measure the intensities of parallel (Ip) and perpendicular (Is) polarized light. Changes in the refractive index close to the surface due to polymer or particle adsorption lead to a change in the ratio of these intensities. The reflectometry signal Ris given by:
p s
R I
I (1.16)
The intensities are proportional to the reflectances RpandRs, respectively, and related to the reflectometry signal Rthrough an unknown instrumental constantC:
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p s
R CR
R (1.17)
The instrumental constant can be eliminated by normalizing signal to its initial valueR
0 as:
0 0 R t R
S t R
(1.18)
This reflectometry signal S t
is directly proportional to the adsorbed mass per unit area according to:
t S t( ) A (1.19)
where
t is the adsorbed mass per unit area at timet, andAis the sensitivity constant.The sensitivity constant is system dependent and one should evaluate the effect of the adsorption on the value of the reflectometry signal in order to quantify the sensitivity constant. For polymers or very small particles, a homogeneous four-slab model can be applied to calculate the effect of the adsorbed mass on the value of the reflectometry signal, with the use of Fresnel equations and Abelès matrix formalism [107, 115]. Every layer on the surface is assumed to be homogeneous and characterized by the thickness and the refractive index of that layer (Figure 1.10). The adsorbed polymer layer is treated as the topmost slab with a certain refractive index nL which can be calculated, assuming the validity of the perfect mixing law, as:
L s
L
n n dn
dc d
(1.20)
where nsis the refractive index of the solvent, dn
dcis the refractive index increment of the adsorbed material, anddLis the thickness of the adsorbed film.
The refractive indices and the thicknesses of each layer in the given multilayered system can be obtained from an ellipsometric measurement and the refractive index increment from a measurement by a differential refractometer. Using the Fresnel equations and Abelès matrix formalism, one obtains a theoretical calibration curve which slope determines the sensitivity constantA. Knowing then all the optical characteristics of the substrate, one can obtain the adsorbed mass.
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Figure 1.10 Schematic representation of the surface structure used for the optical modeling. Polymers are adsorbed on the Si wafer with a SiO2 layer. Each layer is considered to be homogeneous and is characterized by its thickness and refractive index.
1.5 Outline of the thesis
Adhesion and mechanics of dendronized polymers adsorbed on solid substrates were investigated by means of surface sensitive techniques, namely atomic force microscopy and reflectometry. The thesis is divided into 7 chapters. The first chapter provides the theoretical background of polymer adhesion, single polymer mechanics and describes the principle of the surface sensitive techniques used.
The second chapter describes the desorption of individual DPs of different generations from various surfaces studied by AFM-based single-molecule force spectroscopy (SMFS). By combining the AFM imaging and force measurements, the effects of surface hydrophobicity, ionic strength, and polymer generation on the adhesion behavior of DPs were investigated at the single-molecule level. Depending on the surface modification, solution conditions and polymer in question, the polymer-surface interactions can be tuned at the molecular level.
The third chapter focuses on the measurement of force-extension profiles of individual DP molecules.
A novel nano-handling technique based on AFM was developed allowing the investigation of the mechanical properties of the polymers on truly single molecule level. This is ensured by coupling of imaging and force measurements in one experiment. One single polymer molecule is hold between the AFM tip and the opposing surface and can be stretched many times back and forth. Using this technique, the force response of individual DPs in electrolyte solution of different ionic strengths was recorded and described by the freely jointed chain model, showing that the mechanical response of DPs can be tuned by the solution composition.
The fourth chapter reports on a SMFS investigation of the stretching response of DPs. Various methods have been proposed to obtain a single molecule response from the pulling experiments, namely nano-handling, classical pulling, and fishing technique. The nano-handling technique identifies a single polymer molecule on the surface by AFM imaging, and this molecule is subsequently probed during the
27
force measurements. The force response can be then directly attributed to a single polymer molecule. The classical pulling and fishing methods, however, do not provide such a direct way to identify if the pulling event originates from a single or several polymer molecules. This chapter demonstrates that not all the methods are suitable to obtain a single molecule stretching response, and this response is for some techniques substrate and solution conditions dependent.
The fifth chapter discusses the polymer physics of DPs adsorbed onto various surfaces. The persistence length of DPs in electrolyte solution of different ionic strengths was obtained from AFM images using the decay of the bond-bond correlation function and the internal end-to-end distances. This investigation showed that the persistence length of DPs increases with increasing generation and decreasing ionic strength. The magnitude of the persistence length strongly varies with the surface, suggesting that the specific polymer-surface interactions affect the molecular conformation.
The sixth chapter extends the study of adsorption of DPs onto a solid substrate. Reflectometry and AFM were employed to investigate the adsorption process of DPs at different salt levels. Combination of these two techniques allowed to follow the adsorption process of DPs in greater detail, to obtain the adsorbed amount, and to explore conformational changes of adsorbed DPs. It was demonstrated that with increasing ionic strength and generation, the adsorbed amount significantly increases. AFM imaging supports well the results obtained from reflectrometry. Furthermore, AFM image analysis shows that the conformation of adsorbed polymer chains is dependent on the surface coverage.
The seventh chapter presents the basic conclusions of the thesis.
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2 Interactions between Individual Charged Dendronized Polymers and Surfaces
*2.1 Introduction
Since their discovery in the eighties, dendtritic architectures continue to capture the imagination of chemists [79, 116-119]. Globular dendrimers consist of several dendrons attached to a central core, and they represent the most widely investigated structures. While dendrimers of lower generation have relatively loose inner structure, higher generations are rather densely packed and uniform, even though this packing may be influenced by changing the solvent quality [117, 120-122]. Dendrimers have unusual properties, for example, their specific viscosity goes through a maximum with increasing molecular mass [123] or their charge may build up in even-odd shell fashion [124]. At higher concentrations, they assemble in liquid- crystalline structures [125, 126]. Dendrimers have been proposed for interesting applications, for example, as gene vectors [127, 128], drug delivery systems [129-131], or catalysts [116, 132, 133].
Dendronized polymers (DP) carry dendrons at each backbone repeat unit and they represent the other important class of dendritic architectures [79, 119]. The properties of these polymers have been studied in lesser detail than dendrimers so far. Their radial density profile resembles the one from dendrimers, whereby crowding occurs at lower generations due to packing constraints [134]. DPs assemble into a variety of structures including fibrils [85], vesicles [135], or liquid crystals [136]. Potential applications of DPs are often exploiting their responsive behavior [82-84, 137-139], and include the fabrication of optical devices [73,
74] or nanoparticles [140], supports for enzymes or nucleic acids [76, 77, 141], sensors for bacteria [75], and other biological uses [75, 78].
The atomic force microscope (AFM) represents a powerful tool to study dendrimers and DPs on surfaces. AFM imaging has shown that adsorbed dendrimers flatten substantially when adsorbed on solid substrates [142-145], and that their swelling degree can be controlled by the nature of the substrate [146]. Dendrimers adsorbe on oppositely charged substrates and form loose monolayers with liquid-like structure [144, 147]. DPs are known to flatten in their adsorbed state, even though the flattening is less extensive than for the dendrimers [81]. Covalent reactions could be induced by AFM nanomanipulation of
* Reprinted with permission from (Grebikova, L.; Maroni, P.; Muresan, L.; Zhang, B. Z.; Schlüter, D. A.;
Borkovec, M. Macromolecules 2013, 46, 3603). Creative Commons Licence (2015) American Chemical Society.
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individual DPs on surfaces [148, 149]. AFM studies also revealed that charged DPs can fold back on themselves to create duplex bundle structures [85, 86].
Mechanical properties of DPs were investigated with the AFM by means of single molecule force spectroscopy [137, 150, 151]. This technique was pioneered by Gaub and coworkers [27, 28] and permits to study the extension of individual polymer chains upon an applied force. One finds that the stiffness of DPs increases with increasing generation, and that their mechanical properties may depend on the type of solvent or the salt level [137, 150].
Force spectroscopy has not yet been used to study interactions of DPs with surfaces. Polymers weakly interacting with surfaces can be peeled from them, and this process leads to characteristic staircase-like force profiles [14, 28, 29, 152, 153]. The peeling force represents a direct measure of the affinity between the polymer and the surface in the solvent in question.
In this article, we report on investigations with AFM of the peeling of individual polymethacrylate- based DPs terminated with amine groups adsorbed to mica, hydrophobized mica, and gold from aqueous electrolyte solutions at pH 4. Under these conditions, all terminal amine groups of the DPs investigated are positively charged. Favorable conditions for peeling of DPs can be best realized on hydrophobized mica, and one finds that the peeling force strongly increases with increasing dendron generation. We also find that the DP of generation 5 peels from surfaces in a much more complex fashion than the lower generations and we argue that this behavior is related to the pearl-necklace structure of this polymer.
2.2 Experimental section
Dendronized Polymers. The attach-to route was used to synthesize polymethacrylate-based DP of different generations (PGn, n = 1–5) terminated with amine groups as described elsewhere [80, 154]. The polydispersity of the side dendrons is less than 1% when expressed as the deviation from the theoretical number of the amine groups per repeat unit.
The molecular weight of the charged PG4 sample used in this study was determined by applying gel permeation chromatography (GPC) to its neutral direct precursor, a PG4 DP in which all terminal amine groups are tert-butyloxycarbonyl (Boc)-protected [80]. This way the use of aqueous GPC could be avoided.
Deprotection of Boc-protected DPs with trifluoroacetic acid proceeds quantitatively so that neither the molecular weight nor the distribution are affected by this mode of operation. The GPC measurements were performed on a PL-GPC 220 instrument with a 2×PL-Gel Mix-B LS column set (2×30 cm) equipped with refractive index (RI), viscometry, and light scattering, using DMF (LiBr, 1 g/L) as eluent at
30
a temperature of 45°C. Universal calibration was carried out using poly(methylmethacrylate) standards in the range of 2.7×103 to 1.5×106 g/mol (Polymer Laboratories Ltd, UK).
Polymers were always dissolved in 10 mM KCl electrolyte solution of pH 4.0 unless noted otherwise.
Some experiments were carried out in 1 mM and 100 mM KCl solutions. Eventual rinsing was done with the same electrolyte solution.
Surfaces. Three types of surfaces were used, namely mica, hydrophobized mica, and gold. High grade mica was obtained from Plano (Wetzlar, Germany). Bare mica surfaces were obtained by cleaving prior to each experiment in air. A hydrophobized mica surface was obtained by vacuum silanization. More precisely, freshly cleaved mica was placed in an evacuated glass desiccator aside a 200 µL drop of dimethoxy(methyl)octylsilane (Sigma Aldrich, Switzerland) for 30 min. Single crystal gold Au(111) surfaces grown on mica were obtained from Phasis (Geneva, Switzerland). The gold surfaces were rinsed with 2% Helmanex solution, washed with pure water, dried in a stream of nitrogen, and ozone cleaned with an UV-ozone cleaner (PSD Pro, Novascan, Ames, USA) in an oxygen enriched atmosphere for 20 minutes. Contact angles of the different surfaces were measured with a home-made video-camera setup and they are summarized in Table 2.1.
After cleaning, the surfaces were immersed in DP solution, and rinsed with electrolyte solution prior to imaging or force measurements. Mica and hydrophobized mica were incubated in the polymer solution for 40 s, while gold surfaces for 3 min. Whether the samples were kept wet or they were dried in between and rewetted had no effect on the AFM results.
Imaging and force spectroscopy. AFM imaging was carried out in AC-mode with a Cypher (Asylum Research, Santa Barbara, CA). Imaging in solution was carried out with Biolever mini cantilevers (BL- AC40TS, Olympus, Japan) with a nominal tip radius smaller than 9 nm and a resonance frequency of 25- 36 kHz. Images were acquired with scan rate of 4.88 Hz, free oscillation amplitude (FOA) of about 20 nm, and a set point corresponding to around 70% of the FOA. The root mean square (RMS) roughness of the bare substrate was determined by imaging 1 µm2 of each samples and they are reported in Table 2.1.
Table 2.1 Properties of the Surfaces Used.
Surface Root Mean Square
Roughness (nm) Contact angle (degrees)
Bare mica 0.07 0
Hydrophobized mica 0.14 12
Gold 0.19 42
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To analyze lateral sizes and the volumes of the adsorbed molecules, a tip erosion algorithm was applied to the recorded images to correct for the tip convolution effect [155]. This procedure requires the tip shape profile, which is obtained by imaging spherical particles in the electrolyte solution. For PG1, gold nanoparticles (Sigma Aldrich) with diameter of about 5 nm were used, while for larger generations polystyrene particles (Nanosphere Size Standard, Thermo Scientific, USA) with a diameter of around 30 nm. These particles were previously adsorbed on mica functionalized with polyethyleneimine of a molecular mass of 25 kg/mol (Polysciences, Eppenheim, Germany). The tip geometry was extracted by applying the erosion algorithm and by assuming that the particles are spherical with a diameter equal to their height, which can be accurately obtained by the AFM image [155]. The volumes of the polymers were calculated with the Gwyddion software freely available at http://gwyddion.net/.
Force spectroscopy measurements on individual DPs were carried out with the Cypher AFM. The Biolever mini cantilevers were silanized overnight in the gas phase in an evacuated container in the presence of (3‐glycidoxypropyl)dimethylethoxysilane (Sigma Aldrich, Switzerland). DPs were first imaged in the electrolyte solution according to the same protocol as described above. Due to tip broadening caused by the silanization process, the images obtained with functionalized tip had usually inferior lateral resolution than those acquired with a bare tip. To avoid thermal drifts during the force measurements, the DP were first continuously imaged with the AFM for about one hour. When the image has stabilized, the piezo excitation of the cantilever was stopped and the tip was placed on a particular location on a selected molecule. Subsequently, a series of 300 approach-redraw force curves was recorded with a sampling rate of 10 kHz, whereby the deflection of the cantilever and the vertical piezo displacement were acquired as a function of time. To preserve the tip functionalization, a deflection set point towards the surface smaller than 30 nm was used. The cantilever spring constants were in the range of 0.04–0.09 N/m as measured through thermal fluctuations in air [156]. The retraction velocity used was 200 nm/s. Retraction force curves were very similar when the velocity was varied in the range of 100–300 nm/s. After these force measurements, the same region was imaged again. The cleanliness of the tip was checked by recording force curves in a part of the surface that was free of adsorbed molecules. A clean tip did not show any single molecule events. When such events were observed, the measurements were discarded. This procedure assures that the force measurements are truly performed at the single-molecule level and allows the detection of eventual lateral rearrangement after external mechanical excitation.
High resolution imaging. High-resolution scanning probe microscopy probes with tungsten spikes on the apex of a silicon tip (Hi’Res-W14/AIBS, µmasch, Tallinn, Estonia) were used for high resolution imaging
