Chain Conformation and Manipulation

1.14

Chain Conformation and Manipulation

A Kiriy and M Stamm, Leibniz-Institut für Polymerforschung Dresden e.V., Dresden, Germany © 2012 Elsevier B.V. All rights reserved.

1.14.1 1.14.2 1.14.3 1.14.4 1.14.5 1.14.6 1.14.7 1.14.8 1.14.9 1.14.10 1.14.11 References

Introduction Chain Conformation PEs at Surfaces Study of Helical Conformations by AFM Conformation of Polymer Stars Motion of Single Molecules Manipulation of Polymer Conformation in Shear Flow Nanomanipulations with AFM Tip Chemical Modification of Single Polymer Molecules Nanodevices from Single Polymer Molecules Conclusions and Outlook

1.14.1 Introduction The chain conformation of polymer in bulk and solution are typically determined by scattering techniques.1,2 The invention of atomic force microscopy (AFM)3 opened a new perspective in the investigation of a large variety of physicochemical properties of very small objects, including single polymer4 and biomole­ cules.5,6 The possibility of manipulating individual entities at nanoscale has always attracted the scientist. It is therefore not surprising that AFM has become a very powerful instrument for performing single-molecule experiments (SMEs) at surfaces: real-time observation and manipulation of single molecules, investigation of their mechanical properties, and even monitor­ ing of chemical and bio-chemical reactions. AFM measurements can be performed at various interfaces including solid–gas, solid–vacuum, and solid–liquid interfaces, thus enabling studies not only of static properties but also of dynamic processes in solution, such as (macro)molecular motions or chemical reactions occurring at surfaces, may be investigated to some extent.7 To date, virtually all knowledge of physical, chemical, or engi­ neering processes has been obtained from bulk measurements that provide the information averaged over a large number of molecules. However, at the level of the individual molecules, the picture would be quite different: individual molecules can be found in states far from the mean population, and their instanta­ neous dynamics are random. In particular, SMEs performed by AFM allow experimenters to access processes by following the behavior of individual molecules at surfaces. Hence, it is possible to measure distributions describing certain molecular properties, characterize the kinetics and thermodynamics of molecular reac­ tions, and observe possible short-living intermediates. The first impressive SMEs were performed with biological objects, such as DNA, proteins, and viruses.8 In particular, SMEs of biopolymer dynamics have led to the discovery that identical molecules exposed to the same conditions follow different paths to a new equilibrium state.9 SMEs with ‘biological’ systems such as DNA,10 ion channels,11 membrane proteins, enzymes,12 and molecular motors13 have already provided important insights into the mole­ cular mechanism of their biological functions. However, single-molecule studies of ‘synthetic’ polymers are still a significantly less explored area of research. To some extent, Polymer Science: A Comprehensive Reference, Volume 1

367 368 370 372 374 374 377 380 382 382 384 384

this is because of technical difficulties that one faces during investigations: synthetic polymers are usually less well defined and often significantly smaller in size than biological objects. At the same time, investigation of synthetic polymers at the single-molecule level would help to study various ‘classical’ but still unresolved problems in polymer physics and chemistry, such as polymer adsorption, coil-to-globule transition (CGT), crystal­ lization, and reactivity. The great potential of AFM was recently expanded beyond imaging. Indeed, AFM allows one to explore various physicochemical properties of nanoscale objects in a quantitative manner and finally to employ the obtained informa­ tion to optimize the properties of functional (nano)materials. A proper immobilization of the macromolecule at the sur­ face is an essential factor for the successful visualization by AFM. The macromolecule must be fixed on the surface to an extent that it does not move due to diffusion during measure­ ments (or that it moves at least with a rate slower than the tip scanning rate at the surface). Moreover, the adhesion between the molecule and the surface has to be stronger than the inter­ action forces between the molecule and the AFM tip during scanning. The use of noncontact or intermittent (e.g., tapping) mode,14 when compared to contact-mode AFM, considerably decreases the magnitude of the tip–sample interactions, grant­ ing an almost noninvasive visualization of soft materials, such as polymer chains and biointerfaces.15 Despite the impressive progress achieved in visualization of single synthetic polymer molecules, many types of polymers cannot be observed by AFM with molecular resolution due to lateral smearing effects. Thus, single molecules of the majority of the ‘usual’ flexible polymers with a persistence length below 5 nm, such as polystyrene, appear on AFM images as hemisphe­ rical structures and resolution of molecular details is hardly possible. On the other hand, polymers revealing a high chain stiffness and sufficiently large-molecular contour length are much better models for AFM SMEs. Thus, AFM was successfully applied to visualize single molecules of intrinsically stiff comb­ like, dendronized, and helical polymers as well as polyelectrolytes (PEs). In this chapter, we will highlight a series of fascinating AFM experiments with single polymer molecules, with a particular emphasis on the study of polymer conforma­ tions at surfaces as well as on mechanical and chemical

doi:10.1016/B978-0-444-53349-4.00015-7

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Chain Conformation and Manipulation

manipulation of single polymer molecules. Many other experi­ ments in particular on biopolymer molecules will not be considered.

1.14.2 Chain Conformation AFM is an ideally suitable tool for the study of conformational properties of sufficiently stiff biological and synthetic polymers. When deposited and tightly adsorbed from highly diluted solu­ tions, such molecules appear on AFM images as isolated wormlike structures. Processing of the images gives useful infor­ mation on the contour length, the curvature, and the end-to-end distance as shown in Figure 1. This enables quantitative analysis of both the local properties (chain configuration and flexibility) and the overall conformation (excluded volume effects and random walk statistics). One has to keep in mind, however, that one can only study the chain conformation after the adsorp­ tion process, where the chain will have changed the solution conformation during or after the adsorption process. In general, such investigations allow the experimental verifi­ cation of theoretical models describing polymer conformations and to compare solution conformations (typically obtained by scattering techniques) with conformations of polymer chains adsorbed on surfaces. The molecular flexibility, that is, resistance to in-plane bending, is characterized by the persistence length. 16–18 Experimentally, one can use two complementary ways to evaluate the persistence length from the images of the chains. The bond correlation function

Θ l

〈R 20〉

Figure 1 Parameters that became accessible upon visualization of single molecules, that is, contour length L, the end-to-end distance 〈R2〉, and the local curvature ρ = dθ/dl. From Sheiko, S.S.; Möller, M. Chem. Rev. 2001, 101, 4099;4 Figure 3.

(a)

(b)

〈cosðΘÞ〉 ¼ e−l=lp gives the average cosine angle between the tangents along the molecule separated by distance l. The characteristic length lp corresponds to the persistence length. Since the method evaluates the local curvature, it can be applied to molecules of either length. The second method is based on the Kratky–Porod formula,19 which depicts the dependence between the end-to-end distance 〈R2〉, the contour length L, and the persistent length as � � lp 〈R20 〉 ¼ 2lp L 1− ð1−e−L=lp Þ L The Kratky–Porod formula may not be applicable for long molecules with L ≫ lp, for which excluded volume interactions should be taken into account. Intramolecular excluded volume effects result from repulsion between segments within the same molecule, which result in an increase of the end-to-end dis­ tance. These effects are particularly strong for 2D systems, which demonstrate an increased density of segments and do not permit chain crossings. Both methods require complete visualization of a statistical ensemble of single molecules in order to determine L, θ, and 〈R2〉. In addition, they assume the observation of molecules in their natural state, in which mole­ cules are not constrained and freely fluctuate around their equilibrium conformation. The concurrent effects of adsorp­ tion, solvent evaporation, and capillary forces can, however, lead to kinetically trapped conformations. The question arises whether and under what conditions an equilibrium 2D con­ formation can be achieved. A pioneering work describing chain conformation at the single-molecule level was published by Rivetti et al.20 in 1996. It was found that depending on the substrate and the solution, the deposition of double-stranded DNA (ds-DNA) from aqu­ eous solution may lead to an ensemble of macromolecules with end-to-end distances corresponding to either equilibrium or nonequilibrium conformations. Figure 2 displays tapping-mode AFM images of ds-DNA molecules adsorbed from the same solution onto mica using three different deposition methods leading to either equilibrated (Figure 2(a)) or kinetically trapped chains (Figures 2(b) and 2(c)). DNA molecules deposited onto untreated, freshly cleaved mica were able to equilibrate on the surface. Qualitatively, the molecules appear extended with very few crossovers even when the surface is crowded (Figure 2(a)). On the other hand, when DNA was deposited on glow-discharged mica (Figure 2(b)) or water-treated (H+-exchanged) mica (Figure 2(c)), the molecules 4 nm

(c)

0 nm

2 μm

2 μm

2 μm

Figure 2 AFM images of the 1258 bp DNA fragment deposited on mica with three different deposition methods (see explanation in the text). From Rivetti, C.; Guthold, M.; Bustamante, C. J. Mol. Biol. 1996, 264, 919;20 Figure 3.

Chain Conformation and Manipulation

appear condensed, with a considerable number of intrinsic crossovers. In these cases, the mean-square end-to-end distance 〈R2〉 is much smaller than the theoretical 〈R2〉2D. Instead, it is close to the value predicted for molecules existing in three dimensions and orthogonally projected onto the surface plane. Therefore, DNA molecules deposited on such treated mica do not equilibrate on the surface but are trapped immediately upon contact. For freely equilibrated chains (Figure 2(a)), the persis­ tence length of 53 nm was determined, which is similar to what was found by scattering methods for ds-DNA in solution. The approach developed in the Bustamante group was later applied in the investigation of other stiff polymers, for example, poly(isocyanodipeptides) (PICs) and bottle brushes.21 PICs represent an important class of extraordinarily stiff synthetic polymers. The intrinsic rigidity of PICs, which is due to hydro­ gen bonds along the main chain of the polymer, permits their visualization by using AFM with a high resolution.22 The statis­ tical analysis of the curvature of isolated polymeric chains of PICs equilibrated in quasi-2D on the basal plane of mica sur­ faces revealed that the chains possess a persistence length Lp of 76 nm (Figure 3). This indicates that these single polymer mole­ cules are very rigid, that is, even more rigid than ds-DNA. This rigidity was attributed to the helical structure of the polymer backbone stabilized by hydrogen bonds developed between the alanine moieties in the side chains.22 While imaging of flexible single linear macromolecules by AFM often suffers from the still limited resolution, cylindrical polymer brushes (CPBs) or ‘molecular bottle-brush’ (a)

macromolecules, where each or most of the monomers of the polymer backbone are grafted by a linear polymer side chain, can nowadays be imaged routinely. The interest in this unique hierarchical polymeric architecture arises from the fact that the extended, wormlike chain conformation enables single macromo­ lecular visualization and manipulation, and that particular behavior in solution and bulk has been observed for such macro­ molecules. If the length of the backbone is significantly longer than that of the side chains, intramolecular excluded volume effects cause the polymer to adopt a cylindrical shape with the backbone polymer in the core from which the side chains emanate radially.23–28 The properties of the side chains, for example, che­ mical structure, dimensions, stiffness, and mutual interactions, were proven to be determining factors for the conformational behavior of the molecules. This opens opportunities to control the properties of the molecules of interest by synthesizing cylind­ rical brushes with predefined side-chain characteristics and, for instance, to adjust these characteristics by external stimuli, such as solvent quality,29 ionic strength (IS),30 pH,31–34 or temperature,35 by monolayer compression,36 and by surface tension variation.37 Responsive materials may be synthesized accordingly. Thus, the interest in cylindrical brushes is due to conformational effects caused by competition of the entropic restoring force of the extended backbone and the repulsive, steric interaction forces between the side chains. Gunari et al.38 studied mechanical properties of single CPBs with polyisopropylacrylamide (PNIPAM) side chains depos­ ited on mica with AFM. Visualization and stretching of

(b)

250 nm

500 nm (a)

40

(b)

(c) 200

100

160

20

10

Occurrences

80 (nm)

Occurrences

30

60 40

0 50

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(nm)

200

250

120 80 40

20 0

369

0

0 0

20

40

60

L (nm)

80

100

–0.75 –0.50 –0.25 0.00 0.25 0.50 0.75 Theta (rad)

Figure 3 Top: AFM images of a PIC sample. White arrows indicate intersections of separate chains and black ones mark segments consisting of intercoiled chains. Bottom: (a) Histograms of the distribution of the contour lengths of PIC obtained from the AFM images. (b) Evolution of the end-to-end distance vs. the contour length. Theoretical function for 2D-equilibrated chains (dotted line), 2D-trapped chains (dashed line), and experimental results (filled line). (c) Distribution of the θi values as determined from the experimental results and the calculated Gaussian function. From Samori, P.; Ecker, C.; Gössl, I.; et al. Macromolecules 2002, 35, 5290–5294;22 Figures 3a,b and 5a–c.

370

Chain Conformation and Manipulation

individual molecules by AFM tip in aqueous solution revealed the semiflexible nature of the cylindrical macromolecules. Imaging of the brushes on mica and inferring Lp from a versus L plot results in an average persistence length of lp·Lp  29 nm, assuming that the chains adopt their equilibrium conformation on the surface. On the other hand, stretching experiments performed by pulling adsorbed molecules by AFM tip suggested that an exact determination of the persis­ tence length using force extension curves is impeded by the contribution of the side-chain elasticity. Modeling of the stretching of the cylindrical brush molecule as a function of the extension of a dual chain (side chain and main chain) explains the frequently observed very low persistence lengths arising from a dominant contribution of the side-chain elasti­ city at small overall contour lengths. Schmidt et al. estimated the ‘true’ persistence length of the cylindrical brush molecule from the intercept of a linear extrapolation of a (Lp)−1/2 versus L−1 plot. By virtue of this procedure, a ‘true’ persistence length of 140 nm for the PNIPAM brush molecules was found, which is by far larger than the value obtained from image analysis. This deviation was attributed to the strong surface–polymer interactions leading to nonequilibrium conformations of the brush molecules on the mica surface.

1.14.3 PEs at Surfaces PEs, which are macromolecules with ionizable groups,39 are another class of polymers suitable for AFM investigations. An increase of entropy due to the release of counterions in polar (aqueous) medium causes ionization of PE molecules. At a high charge density, PE chains display an extended coil conformation due to the electrostatic repulsion between charged monomer units so that observation of fine molecular details with AFM becomes possible. PEs undergo diverse conformational transi­ tions responding to change of environment (IS, pH, condensation agents, temperature, concentration) driven by the interplay between attractive short-range van der Waals and repul­ sive long-range Coulomb interactions. Conformational changes of bio-PE molecules, such as proteins and DNA, are involved in many natural phenomena in living systems so that these pro­ cesses are of a vital importance. The response of PE molecules to changes in pH and IS of aqueous solutions has been intensively explored for engineering man-made stimuli-responsive polymer systems. Several properties of different materials were switched and tuned based on pH- and salt-responsive behavior of PEs: wetting of mixed polymer brushes,40 adhesion,41 colloidal sta­ bility, protein adsorption,42 transport of drugs and ions in PE gels,43 and microfluidic channels,44 actuation,45 and templating single PE molecules for the fabrication of metallic nanoparticles of various sizes and shapes.46 Studies of the conformational transitions of PE molecules at the single-molecule level play an important role in the understanding of PE behavior in the above-mentioned materials and systems. The CGT has been studied theoretically, by simulations and experimentally. The behavior of highly charged PE molecules in diluted solutions and at the surfaces is well understood and studied using light, X-ray, and small-angle neutron scattering (SANS). Originally driven by a purely fundamental interest in the CGT postulated by Stockmayer47 and de Gennes,48 it has meanwhile gained a considerable technological relevance. The

collapse of coils to compact spheres was proven for the first time in 1980 by Sun et al.49 Successively, several features of intermediate structures have been addressed. Chu et al.50 iden­ tified a two-stage process with the first stage being attributed to a nucleation and growth of beads along the chains, which further grow and merge during a second stage. It is well known that at high charge density PE chains display an extended coil conformation due to the electrostatic repulsion between charged monomer units. Decrease of the charge density results in a collapse transition of PE chains to a globule confor­ mation due to the short-range interactions. A change of PE conformations in a controlled environment and, particularly, CGT phenomena attract continuously enhanced interest due to their importance in industry and nature. Thus, gelation of PE in water and reversible swelling or shrinking of PE gels responding to external stimuli are interesting applications of the CGT phenomenon. CGT is considered to be a complicated process where the transition character depends on chain stiffness and specific inter­ actions in the system.51 Traditionally, polymer science considers CGT for flexible polymer chains as a gradual process that is associated with a second-order phase transition,52 while for stiff polymers the theory suggests53 a sharp first-order phase transition. In contrast to the theoretical predictions, many experimental results have indicated a smooth continuous char­ acter of CGT for different flexible and stiff polymer chains. SMEs with stiff DNA molecules have shown that an individual chain exhibits a first-order phase transition between an elongated coil and a spherical or ‘ordered’ toroidal54 globule, while this transition appears continuous in the ensemble. In contrast, DNA modified by synthetic polymers undergoes a continuous second-order phase transition through a set of partially collapsed ‘disordered’ intrasegregated conformations.55 That displays the general tendency for polymers where the highly cooperative all-or-non character of the collapse transitions usually is present for ordered folding reactions or very stiff molecules, where in both cases the density of the compact state is much larger than that of the coil.56 The mechanism of CGT for synthetic flexible PE on the single-molecule level is still not fully resolved. Theoretical postulations for the molecular details of confor­ mational transition of PEs have been first presented by Kantor and Kadar57 and Dobrynin et al.58 who transferred the concept of the shape instability of charged oil droplets to collapsing organic polyampholytes and homogeneously charged PE coils in water, respectively. Unlike the oil droplets, however, the resulting subdroplets of partially collapsed charged PE coils remain interconnected by string-like coil segments. The reverse process, that is, the expansion of such collapsed PEs, leads to a cascade of transition states, which resemble necklace-like struc­ tures. These transition states bear only partial similarity to the electrically neutral intermediates59 mentioned above. Contrary to the latter, the electrostatics of PE intermediates aligns the splitting beads equidistantly along a straight line if no additional salt is present in solution. Screening of electrostatic repulsions due to the added salt may disturb this alignment of beads. It is also noteworthy that the electrostatic nature of the shrinking coils may stabilize intermediates and thus makes its investiga­ tion easier than in the case of neutral polymers. It could be revealed by further theoretical investigations and simulations that a subtle interplay of solvent quality for the hydrophobic backbone, of polymer concentration, and of the degree of

Chain Conformation and Manipulation

electrical charging is required to generate such necklace-like intermediates.60 In the necklace regime, large fluctuation of the bead size and number of beads per chain occur within a sam­ ple.61 Those fluctuations occur with time but are also present between different molecules in the ensemble. If all other parameters are appropriately fixed, coil shrinking can be induced by discharging the molecule. The charge of a PE molecule can be controlled by counterion condensation, by complex bonding of specifically interacting counterions, by variation of the fraction of chargeable groups in a copolymer, or by changing the solvent quality for the polymer. A survey of these contributions has been given recently.62 Many of the experimental reports on PE shrinking support the model of a necklace-like chain conformation. Experiments that are considered to reveal the most direct indications for pearl necklace structures are small-angle scattering curves from single chains and AFM images of isolated coils. Aseyev et al.63 induced the shrinking of PEs in salt-free water by addi­ tion of acetone and performed SANS experiments with collapsed chains. By comparing the overall size of PE chains with the behavior of the scattering curves at high q-values, they found an indication for a string of three to four beads. Schweins et al.64 investigated the collapse of polyacrylic acid, sodium salt (NaPA), induced by divalent alkaline earth cations. AFM makes accessible the conformation of adsorbed PE coils. During adsorption, the conformation of long polymers is inevi­ tably modified by the transition from three to two dimensions.65 Loss of one degree of freedom reduces the number of possible configurations that the molecule can access within the range of the thermal energy. In principle, three distinct cases of molecule– surface interactions can be postulated: (1) the molecules freely equilibrate on the surface to a two-dimensional (2D) conforma­ tion before they are captured in a particular conformation; (2) the PE coils adsorb irreversibly whereby the interaction with the substrate changes the polymer conformations, leading to only their partial local equilibration; or (3) the molecules adhere to the substrate without being equilibrated, and the resulting con­ formation resembles a projection of the actual three-dimensional (3D) conformation onto the surface. The latter scenario corre­ sponds to kinetic trapping of the molecules onto the surface. In light of these three distinct cases, the question arises of how much the conformation of adsorbed molecules reflects the respective conformation in solution before adsorption. Recently, AFM has been used for the study of the reconfor­ mation of poly(2-vinylpyridine) (P2VP).66 It was shown that positively charged polymer molecules adsorb strongly onto negatively charged mica leading to a projection of the actual 3D conformation onto the surface. In particular, at pH 2.0 in

(a)

371

aqueous solution P2VP is highly charged, and adsorbed mole­ cules appear in AFM images as wormlike chains with the average thickness of about 0.2 nm (Figure 4). At pH 3.5 P2VP (NaCl 0.02 mol l−1) molecules appear to be much more strongly coiled chains resembling a deformed globule (Figure 4(b)) with an average height of 0.35 nm. The data suggest that a decrease of the fraction of charged monomers affects the transition to necklace-like globular conformation. These globule conforma­ tions resemble dumbbell (Figure 4(c)) and trimbell structures predicted from simulations. At pH 3.5 and NaCl concentration 1.0 mol l−1, the chains undergo a transition to still more compact globule conformations (Figure 4(d)) with the average height of the structures of 1.4 nm. In another work, Kiriy et al.67 investigated chain shrinking for poly(methacryloyloxyethyl dimethylbenzylammonium chloride) by addition of Na3PO4 as a low-molecular-weight salt, which screens intermolecular repulsions. They succeeded to image a cascade of transition states with necklace shape on mica substrates. The number of beads and the length of the interconnecting strings decrease, while the size of the beads increases with increasing salt concentration in the preceding solution state (Figure 5). In a closely related experiment, (a)

(b)

100 nm

100 nm

(c)

(d)

50 nm

50 nm

Figure 5 AFM images of poly(methacryloyloxyethyl dimethylbenzylam­ monium chloride) single molecules deposited from aqueous solutions: reference, no salt (a) and with added Na3PO4 (4.2 mM (b), 6 mM (c), 8.4 mM (d)). From Kiriy, A.; Gorodyska, G.; Minko, S.; et al. J. Am. Chem. Soc. 2002, 124, 13454–13462;67 Figures 7a,be,f.

(d)

(b)

(c)

Figure 4 AFM image of P2VP adsorbed on mica (a) at pH 2.0; (b) at pH 3.5 and NaCl 0.02 mol l−1; (c) inset, zoom of the image (b); (d) at pH 3.5, NaCl 1.0 mol l−1. From Minko, S.; Kiriy, A.; Gorodyska, G.; Stamm, M. J. Am. Chem. Soc. 2002, 124, 3218–3219;66 Figure 2.

Chain Conformation and Manipulation

(a)

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50 nm (b)

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≥5 4

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NaCl l = 0.5 M

NaCl l = 4.5 M pH 3

(e)

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Na2SO4 l = 0.25 M

Na2SO4 l = 4.5 M pH 3

0

pH 3

Chains

Kirwan et al.68 observed essentially the same trend with poly (vinylamine). In the latter experiment, a direct discharge of the respective poly(vinylammonium) salt was achieved by decreasing the pH. It is noteworthy that all captured necklace-like structures were closer to an arrangement of beads along a semiflexible chain than to an equidistant placement of beads along a straight line (stretched necklace). Recently, Lages et al.69 investigated the behavior of longchain sodium polyacrylate polymers in dilute aqueous solution in the presence of Ca2+ ions. To reveal details of this shrinking process, the conformational changes in response to the addition of alkaline earth cations at two different temperatures were studied by means of light and neutron scattering and by AFM on the same samples. The scattering curves from the intermediates were interpreted by a pearl necklace model, which includes a low amount of pearls per polymer separated at 80 nm from each other. AFM investigations of adsorbed chains confirm the drastic conformational changes inferred to the system with temperature increase and formation of pearl-necklace-like intermediates. The results are considered to be one of the rare direct evidences for a pearl-necklace-like intermediate along the CGT of PE chains. In the above considered works, PE conformations were kine­ tically frozen upon a rapid adsorption onto oppositely charged surfaces and further AFM investigations were performed in the dry state. Roiter et al.70,71 studied the structure of the PE coils using in situ AFM experiments in aqueous solutions in a liquid cell. They studied the behavior of hydrophobic PE chains adsorbed at the solid–liquid interface in the full range of possi­ ble salt concentrations. They found that (1) in dilute salt solution, PE chains possess an extended coil conformation visualized as adsorbed 2D-equilibrated coils; (2) in a moderate salt concentration range, the polymer coils shrink and approach the dimensions of polymer coils under θ-conditions and the chains are visualized as adsorbed 3D-projected coils; (3) at high salt concentrations, the polymer coils reexpand and the molecules are visualized as 2D-equilibrated extended coils; how­ ever, (4) reexpansion is limited in the presence of multivalent counterions, presumably due to the bridging of the polymer coils by the counterions. This interesting behavior of P2VP chains was examined using statistical analysis of 2D-equilibrated versus 3D-projected chains. It was shown that the adsorbed polymer chains may occupy either the conformation of the 2D-equilibrated coils, when the interaction with the substrate is sufficiently strong to force the formation of 2D coils or 3D-projected coils (whose conformation reflects the projection of the 3D coil in solution on the solid substrate). In the latter case, the 2D equilibration is limited by strong intrachain forces. The red and blue dash-dot curves in Figures 6(b) and 6(c) reflect the expected 〈r2〉1/2 values for poly­ mer chains of the same degree of polymerization in 2D-equilibrated and 3D-projected states, respectively. These values were estimated from the experimental values of contour length and persistent length of the examined chains. The data show that P2VP chains occupied 2D-equilibrated conformations at low (< 0.01 M) and at very high (> 1 M) IS (for multivalent counterions). In the intermediate range of salt concentrations (0.1 M< IS < 1 M), hydrophobic interactions and ion bridging sta­ bilized 3D-projected coils. The 3D conformation manifested through the increased number of superposed segments in Figures 6(c), 6(e), and 6(f). For the high concentrations

nm

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pH 3

Figure 6 Dimensions of adsorbed P2VP molecules vs. IS and Debye length (λD) in salt solutions: 〈s2〉1/2 (a); 〈r2〉1/2 in NaCl (b) and Na2SO4 (c) solutions at pH 3.0. The experimental data were averaged for about 200 individual molecules. Red dash and blue dash-dot lines (b–c) display estimated 〈r2〉1/2 values for 2D-equilibrated and 3D-projected chains, respectively. From Roiter, Y.; Trotsenko, O.; Tokarev, V.; Minko, S. J. Am. Chem. Soc. 2010, 132, 13660–13662;71 Figure 1.

(IS >1 M) of divalent counterions, the P2VP coils retained the 3D-projected state (Figure 7). Thus, multivalent counterions sta­ bilize 3D coils at high salt concentrations. Multivalent anions, which are strongly correlated at the PE backbone, cross-link the PE (effect of bridging), thereby preventing the complete reexpan­ sion of polymer chains. The results are in accord with simulations and theory,72 except with regard to the strong reexpansion in the presence of multivalent counterions. The latter can be explained by strong van der Waals interactions of the pyridine ring with mica. These interactions lead to a near full recovery of the 2D-equilibrated coil conformation in the adsorbed P2VP chains.

1.14.4 Study of Helical Conformations by AFM During the last decade, a number of synthetic helical polymers were visualized by AFM. The design and synthesis of helical polymers with a controlled helix sense have attracted signifi­ cant attention, due to the inspiration by biological helices and possible applications in chiral materials for the sensing and separation of enantiomers and enantioselective catalysis. However, the determination of helical structures, in particular, the helical pitch and helical sense, is still very difficult. The direct observation of helical polymers by AFM is one of the most promising methods to determine the helical structures.73–77 Despite recent progress, at this stage there is still very little understanding about how chiral molecules self-assemble onto nonchiral surfaces, which is a key point for the chirality to be produced at the supramolecular level in the solid state. Helicity can be induced at the single-chain level by placing chiral groups and/or by steric constraints along the chains. This is the case for instance with poly(phenylacety­ lene)s, which adopt a helical conformation, provided that chiral pendant groups are present.78 With nonchiral substitu­ ents, a one-handed helicity can also be obtained using small, optically active molecules capable of complexing the polymer chains, as suggested by Sakurai et al.79 They also showed that

Chain Conformation and Manipulation

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Figure 7 AFM images of P2VP molecules under salt-free conditions at pH 4.8 (a) and 3.0 (b) and at the moderate to high values of IS

(shown in images) in NaCl (c, d) and Na2SO4 (e, f) solutions. From Roiter, Y.; Trotsenko, O.; Tokarev, V.; Minko, S. J. Am. Chem. Soc. 2010, 132, 13660–

13662;71 Figure 2.

rigid-rod helical poly(phenylacetylene)s bearing L- or D-alanine residues self-assemble into ordered hierarchical 2D crystals under exposure of solvent vapors on highly oriented pyrolytic graphite (HOPG) (Figure 8).80 It was shown that flat mono­ layers immediately and epitaxially form on the basal plane of graphite, on which rodlike helical polyacetylenes further self-assemble into 2D helix bundles with controlled helicity upon exposure to organic solvents. High-resolution AFM ima­ ging revealed their helical conformations in the 2D crystals and enabled the determination of molecular packing, helical pitch, and handedness. These values agree well with those determined by X-ray diffraction of the oriented liquid crystal­ line polymer films. The stereocomplex formed from complementary strands of isotactic and syndiotactic poly(methyl methacrylate)s (it- and st-PMMAs) with an it:st-stoichiometry of 1:2 represents another class of unique, polymer-based helical supramolecules suitable for AFM studies. Although this stereocomplex has been known for half a century, the molecular basis of the structure and the mechanism of complex formation are still under debate. In 1989, Schomaker and Challa81 proposed a double-stranded helix model for the stereocomplex based on the wide angle Xray scattering (WAXS) analysis of the stretched fiber, which is composed of a 91 it-PMMA helix (nine repeating MMA units per turn) surrounded by an 181 st-PMMA helix, resulting in a double-stranded helix with a helical pitch of 1.84 nm. To verify the structure of the PMMA stereocomplex by the AFM techni­ que, Kumaki et al.82 prepared a mixed monolayer of it- and

st-PMMA chains spread on a water surface, compressed it using the Langmuir–Blodget technique and transferred the layer on atomically flat mica. The AFM phase image (Figure 8(b)) revealed well-defined helix-bundle-like structures, which can be further resolved into individual chains with periodic oblique stripes (yellow arrows) having a pitch of 0.92 nm which were tilted either counterclockwise or clockwise along the stereocom­ plex main chain (pink lines). These results indicated that the stereocomplex is indeed a helical polymer-based supramolecule. The chain–chain distance (2.4 nm) estimated from the AFM images was in good agreement with the width of the proposed double-stranded helix model ( 2.4 nm). However, the observed helical pitch (0.92 nm) was equal to half the helical pitch of the double-stranded helix model (1.84 nm). On the basis of the AFM results, in particular, the helical pitch (0.92 nm) of the stereocomplex, combined with the fact that the stereocomplex quantitatively forms at the stoichiometric ratio of it- to st-PMMAs = 1/2, Kumaki et al. proposed a triple-stranded helix model (Figure 8(b)), in which a double-stranded helix composed of two 91 it-PMMA helices with a 1.84 nm helical pitch is surrounded by an 181 st-PMMA helix with the observed 0.92 nm helical pitch. To conclude, the results obtained with helical polymer structures at surfaces pave the way toward new approaches not only for the construction of new chiral materials, such as chiral selectors and catalysts, but also for the rational design of novel switchable chiral surfaces based on inversion of helicity of macromolecules.83–85

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Figure 8 (a) AFM phase image of a monolayer of an it- and st-PMMA mixture (it/st = 1:2) deposited on mica at 10 mN m−1. (b) Magnified image of the area indicated by the yellow square in (a); the pink lines represent the main-chain axes of the stereocomplex; the yellow arrows indicate the antipodal oblique pendant helical arrangements with respect to the main-chain axes. (c) Typical Fourier transform of a section of (a). From Kumaki, J.; Kawauchi, T.; Okoshi, K.; et al. Angew. Chem. Int. Ed. 2007, 46, 5348;82 Figure 3.

1.14.5 Conformation of Polymer Stars AFM was also shown to be a powerful tool for investigation of more complex polymer architectures at surfaces. Thus, Stamm et al. used AFM to investigate conformationals of star-shaped polymers at the single-molecule level. In particular, they found that polystyrene/poly(2-vinylpyridine) (PS7–P2VP7) heteroarm star block copolymers undergo diverse conformational transi­ tions responding to external stimuli.86 At concentrations below 0.01 g l−1, the star PS7–P2VP7 exists in near molecularly dis­ solved state in both selective (acidic water, toluene) and common good (chloroform, tetrahydrofuran) solvents. In the latter case, the obtained structures on mica show that a ‘Janus’­ like segregated structure is likely to exist in chloroform.87 In acidic conditions, PS7–P2VP7 forms either unimolecular or mul­ timolecular micelles depending on concentration, pH, or IS. The core of the micelles is constituted of collapsed PS arms sur­ rounded by protonated P2VP shell (Figure 9). The star PS7–P2VP7 undergoes inverse intramolecular segregation upon addition of toluene. In this case, the inverse unimolecular micelles are constituted of the P2VP dense core and the PS swollen shell (Figure 9). The transition between those two inverse types of micelles is strongly modified by interactions with the mica substrate. The micelles deposited onto mica from acidic water are trapped via P2VP extended arms. Upon treatment of the trapped micelles with toluene, the PS core is swollen and PS arms gradually adopt an extended conformation,

whereas P2VP trapped arms retain their extended conformation due to the strong interaction with the mica substrate. The obtained flattened structures present unique conformations that are not expected to exist in any solvent and could not be obtained upon simple adsorption procedures (Figures 9(f)–9(i)).

1.14.6 Motion of Single Molecules For successful AFM imaging, polymer molecules should be strongly fixed on surfaces to avoid molecular diffusion during measurements. However, the attractive interactions between the molecule and the surface should be reduced to a critical level if one is interested in probing molecular dynamics in a real time. As described in previous paragraphs, in many cases PE molecules adsorb strongly to oppositely charged surfaces. An efficient way was found to induce reconformation and equilibration of noncharged chains on surfaces via exposure of the sample to vapors of appropriate solvents. In particular, Gallyamov et al.88 reported AFM real-time visualization of conformational changes of single linear P2VP chains adsorbed on mica in a controlled vapor environment. Thus, after deposi­ tion on mica from a solution in chloroform, the P2VP molecules adopted a partially extended conformation (Figure 10(a)). However, the polymer chains on the substrate extended even further when the sample was exposed to water vapor (see Figures 10(b) and 10(c)). The extension was a rather slow

Chain Conformation and Manipulation

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Figure 9 (a) Star-shaped PS7–P2VP7 block copolymer. AFM images and schematic representation of PS7–P2VP7 unimers adsorbed on mica from acidic aqueous solution (b–c) and from toluene (d–e); and adsorbed from acidic aqueous solution and then placed in toluene for 30 min (f) or for 5 h (g). (h–i) Schematic representation of the reconformation. From Gorodyska, A.; Kiriy, A.; Minko, S.; et al. Nano Lett. 2003, 3, 365.86; Figure 1a,c; from toluene (d-e), Kiriy, A.; Gorodyska, A.; Minko, S.; et al. Macromolecules 2003, 36, 8704–8711;87 Figure 7.

process, which was easily followed by recording subsequent AFM images, that is, in real time. Measured height values for the extended molecules were in the range of 0.15–0.4 nm above the substrate level. When the sample chamber was purged with dry nitrogen, the humid atmosphere was replaced, and the macromolecules became fully immobilized retaining their conformation, that is, the conformation was ‘frozen in’ at the vapor-free environment. Exposure of the macromolecules to ethanol-saturated vapor induced a reverse transformation; that is, a coil-to-globule con­ formational transition to a compacted state was initiated (see Figures 10(d) and 10(e)). When exposed to the ethanol vapor, the molecules first increased in thickness (this was indicative of some solvent uptake) and then transformed into compact, round particles (globules) with a height of 1.5–3 nm. The compacted molecules can be unfolded again by exposure to water vapor (see Figures 10(f)–10(l)). The real-time observation shows that the extension of the polymer chains occurs in a continuous process. Individual P2VP strands ‘grow out’ from the globules like sprouts from grains. The spreading process has a slightly fluctuating nature that can be clearly seen by following a series of AFM images recorded within short intervals (10 min). The molecules underwent small random shifts, occasional contractions fol­ lowed again by reextensions. It was found that extension of molecules ceases after exposure of the sample for 10 h in water vapor, indicating conformational equilibration. The dimensions of the polymer chains equilibrated in water vapor were described by the 2D SAW (self-avoiding walk) model. The observed contraction dynamics of the P2VP chains on mica in ethanol vapor was explained in the framework of the ‘spreading’ concept. The amphiphilic ethanol solvent mole­ cules exhibit a higher interfacial activity as compared to the polymer molecules. Therefore, coadsorbtion of an ethanol

layer can reverse the spreading of P2VP on a mica surface. That is, the ‘surfactant-like’ nature of the amphiphilic ethanol molecules manifests itself in the effective removal of the poly­ mer chains from the substrate, and the P2VP strands are forced to minimize their surface contacts and to adopt a compacted conformation. The collapse is further promoted by the fact that the P2VP chains are solubilized by ethanol molecules, as is observed by an apparent increase in diameter of the chains. As a consequence, the interaction of the pyridine subunits with mica gets shielded. In a related work, Gallyamov et al.89 reported the in situ AFM observations of reversible conformational transitions of more rigid and thick poly(methacrylate)-graft-poly(n-butylacrylate) (PMA-g-PnBA) brush molecules on mica between compact globule conformations under ethanol vapor and elongated ones under humid air. Recently, Kumaki et al.90 showed that single high­ molecular-weight poly(methyl methacrylate) chains adsorbed on mica in a humid environment diffuse according to a ‘repta­ tional’ motion, that is, along the chain axis (like a caterpillar; Figure 11). Lowering the relative humidity (i.e., the thickness of the water layer on mica), the diffusion coefficient of the chains decreases (Figure 11). It is worth noting that the risk that the AFM tip could induce these ‘reptational’ movements is under debate, but arguments quite convincingly rule out this possibility. An ability of adsorbed macromolecules to reconformate on surfaces was further utilized in an elegant way to induce a direc­ tional movement of single molecules. In particular, the idea of inducing the motion of nano-objects by means of periodic response of a polymer system to cyclic variation of the vapor environment was verified.91 A theoretical basis for these exp­ eriments was recently developed by Perelstein et al.92 They proposed a prototype of a molecular motor that has a ‘simple’

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Figure 10 Sequence of AFM images demonstrating step-by-step the different stages of conformational transitions for individual P2VP polymer chains on mica exposed to the vapors saturated with humidified ethanol (80 vol.%) and water. Three individual macromolecules are marked by colored outlines in the successive images. (a) Initial image of the partially extended molecules as deposited and dried in N2. (b, c) Further extension of the polymer chains in water vapor – the AFM images obtained 1 h (b) and 2 h (c) after injection of water into the environmental chamber. (d, e) Exposure to ethanol vapor transferred the macromolecules to a compacted state – the images are recorded 15 min (d) and 30 min (e) after the ethanol injection. (f–l) Images again demonstrating the unfolding dynamics of the same chains in water-saturated atmosphere 15 min (f), 1 h (g), 2 h (h), 3 h (i), 5 h (j), 7 h (k), and 10 h (l) after injection of water into the chamber. From Gallyamov, M.O.; Khokhlov, A.R.; Möller, M. Macromol. Rapid Commun. 2005, 26, 456–460;88 Figure 1.

internal structure. Particularly, computer simulations predicted that a single diblock copolymer chain comprising ‘simple’ monomer units is able to perform directed reptational motion on being adsorbed on a structured solid surface. From the physical viewpoint, such directional motion is possible if the following three conditions are satisfied: (1) the system gets external energy that can be dissipated, (2) the adsorbed molecule has an anisotropic molecular friction, and (3) the surface provides direction of the motion (‘track’). This situation can physically be realized, if, for example, one of the blocks contains photosensitive groups whose interactions can be controlled by light. Another possibility to induce collapse and readsorption comprises an exposure of the molecule to different vapors.

Relying on the periodical variation of the number of adsorbed monomer units of one of the blocks and, therefore, of the friction force, one can expect fulfillment of the second condition. Finally, the third condition is provided by the structure of the surface that is patterned in stripes. Recently, Sakurai et al.,77 going along these lines, demonstrated that several successive vapor-induced collapse– decollapse cycles of single brush-like macromolecules resulted in lateral shifts of the macromolecules integrally on the substrate. On flat, structureless substrates (e.g., mica or silicon), such lateral shifts were quite randomly oriented. According to the theoretical predictions, the following two next steps should be implemen­ ted in order to develop an artificial molecular walker. The first

Chain Conformation and Manipulation

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Figure 11 Movements of it-PMMA chains on mica under high humidity. (a) AFM image of it-PMMA chains on mica deposited by Langmuir Blodgett (LB) technique (time: 0 min; scale: 484–533 nm; scan direction: left to right). Each chain is indicated by a top (white: freely moving chains; light blue: anchored chains). The trajectories of the center of mass of the chains are shown by blue lines from 0 to 82.5 min in 71% relative humidity (RH) and by red lines from 82.5 to 136.5 min in 54% RH. (b) Time lapse of the chain shapes for chains (g, j, h, n) in Figure 11(a). The chain images are superimposed by changing the color from blue, orange, yellow, pink, and green with time. From Kumaki, J.; Kawauchi, T.; Yashima, E. Macromolecules 2006, 39, 1209–1215;90 Figure 1a,c.

step would be to fix a single direction for the macromolecular movement, that is, to switch from 2D random lateral shifts to one-dimensional (1D) random shifts. The second step would be to introduce some asymmetry into the molecular structure of a prototype of the molecular walker. In such a case, one may expect to observe really directional motion along one fixed direction. Gallyamov et al. studied the motion of diblock bottle-brush-like macromolecules on a mica substrate with friction-deposited highly oriented poly(tetrafluoroethylene) (PTFE) nanostripes. Such nanostripes have been supposed to serve as nanotrails for molecular motion. The macromolecules are characterized by intrinsic asymmetry due to their diblock structure. The motion was induced by repetitive change of the vapor environment (cyclic exposure of the adsorbed macromo­ lecules to ethanol and water vapor). A peculiar twisting motion of diblock copolymers precollapsed in ethanol vapor was visua­ lized by AFM during their subsequent spreading in water vapor. The intrinsic asymmetry of the diblock macromolecules has been considered to be the reason for such twisting. It was found that some of the macromolecules demonstrated a certain tendency to orient along the PTFE stripes, and some of the oriented ones have moved occasionally in a directed manner along the trail. However, it has been difficult to reliably record such directed motion at the single-molecule level due to some mobility of the PTFE nanotrails themselves in the changing vapor environ­ ment. Nevertheless, these results might be considered as a good step toward development of artificial nanomotors.

1.14.7 Manipulation of Polymer Conformation in Shear Flow In most cases, adsorption of flexible polymers leads to quite coiled surface structures that prohibit AFM imaging with mole­ cular resolution. The imaging is significantly facilitated if polymer polymers are deposited in fully stretched

conformation. In addition, determination of the contour length and the molecular weight distribution is straightforward for such fully stretched chains. This is especially important when standard methods of molecular weight measurement are hardly applicable, such as in the case of PEs or for molecules with unknown or complex composition. On the other hand, synthetic and biological macromolecules nowadays become important and versatile building blocks for future molecular electronics.93 The use of single polymer molecules as templates constitutes a highly promising strategy to generate nanoparti­ cles with desired size, shape, location, and with specific properties. For nanotechnological and sensor applications, it is frequently necessary to deposit single polymer molecules in the stretched conformation, for instance, to bridge microelec­ trodes and to construct nanodevices on their basis. Polystyrene sulfonic acid (PSA)94 and some other polyanions (PAs)95 have been immobilized in the stretched conformation by co-deposition with long-chain aliphatic amines (AAs) onto HOPG. Complexation of the negatively charged PAs with the oppositely charged AAs (in water) leads to the formation of relatively thick hydrophobic fibrillar structures. The PA chains align themselves along the AA lamellas adsorbed on the basal plane of the crystalline graphite. These methods, however, are not universal as their applicability is restricted to specific crystal­ line and high surface energy substrates, such as mica or HOPG. In principle, linear macromolecules can be stretched and aligned with the assistance of external forces such as centrifugal or capillary forces and electric or shear fields. This, however, demands that the macromolecule/substrate interactions are accurately balanced. Should they be too strong, the molecules are trapped in their solution conformations. In the reverse case, the adsorption is not feasible. In the ideal situation, the mole­ cules are adsorbed onto the substrate weakly enough that sliding along the flowing direction can still occur. Molecular combing is a well-known approach to stretch and align PE molecules onto solid surfaces.96,97 The first molecular combing

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Chain Conformation and Manipulation

work to achieve a moving meniscus was accomplished by slid­ ing two pieces of glass with a drop of DNA solution sandwiched between them.83 Combing techniques were also applied for the manipulation of intrinsically stiff synthetic polymers. For example, Otten et al.98 have reported on the stretching of extremely long (up to 12 μm) polyisocyanate molecules on HOPG, prepared via acid-catalyzed polymeriza­ tion. Such long stiff PIC strands were considered to be ideal candidates for exploring the effects of hydrodynamic forces and long range (i.e., excluded volume) and short range inter­ actions on the adsorbed chains orientations at different length scales. It was found that single PIC chains on mica exhibit different types of alignment on different length scales, when deposited relatively quickly by drop-casting or spin-coating. The alignment is governed by the interplay between the hydro­ dynamic flow strength, the macromolecular chain length, and the surface recognition. On tens of nanometers scale, the chains recognize the threefold symmetry of the crystalline surface, which leads to oriented chains with a directional persistence of 114 nm. On the micrometer scale, that is, the scale of the contour length, hydrodynamic flow stretches the chains. In another work, shear-induced stretching of relatively flexible positively charged poly[methacryloyloxyethyl-dimethyl-(3­ pyrrol-1-yl-propyl)ammonium bromide] (PME-D-AB) chains adsorbed on a planar substrate (mica) was explored by AFM and Brownian dynamics computer simulations by Stamm’s and Löwen’s groups.99 Considerably stretched polymer chains along the shear flow with a wealth of different individual shapes were found (Figure 12). The asymmetry of the polymer size distribution characterized by the skewness was in good agreement between simulation and experiment under the assumption that the shear gradient

z

Flow direction

x (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

. (a) γτ = 10

. (b) γτ = 50

(c) Region II

Figure 12 (a, b) Typical simulation snapshots of polymer chains under linear shear flow in x-direction. The surface normal is parallel to the shear gradient direction. Here the left column (a) shows configurations for lower and higher shear rates; column (c) shows the experimental chain images obtained at the higher shear rate. From He, G.-L.; Messina, R.; Löwen, H.; et al. Soft Matter 2009, 5, 3014–3017;99 Figure 2.

is normal to the wall. The main conclusion is that the polymer configurations found in the experiments are vastly different from projected 3D bulk configurations but consistent with strongly adsorbed ones under the steady-state condition of an imposed linear shear gradient normal to the wall. This implies that only the late-stage process of drying leads to a simple projection of adsorbed configurations. For increasing shear rates, a consider­ able proportion of stretched chain configurations was found, which are characterized by an increasing (positive) skewness of the chain size distribution function. This behavior is also repro­ duced by Brownian dynamics computer simulations if the shear gradient direction is perpendicular to the wall, but not for the situation where the shear vorticity is normal to the wall. Bocharova et al.100 developed a simple and efficient proce­ dure to tune interaction forces between PE molecules and mica surface so that the conformation and orientation of single mica-adsorbed polycation molecules can be controlled. In par­ ticular, they found that positively charged macromolecules codeposited with octylamine (OA) onto mica appear in a con­ siderably more stretched conformation compared to adsorption onto untreated mica. Furthermore, the molecular thickness is considerably larger whenever the macromolecules are codeposited with OA, which indicates a change in the local conformations of the chains and the orientation of their side groups with respect to the substrate. These observations have been explained by the formation of an ultrathin liquid-like film of OA on the mica, which decreases the surface energy, weakens the interactions of the individual macromolecules with the sur­ face, and allows them to be stretched. Since the contour length and molar mass of the stretched macromolecules can be directly measured, this method is a useful analytical tool (Figure 13). The increase in the molecular height in the case of codeposition with OA drastically improves the molecular resolution, which makes even ultrathin polycations detectable and thus extends significantly the range of polymers that can be used in SMEs. Demidenok et al.101 further developed the molecular combing approach and demonstrated an efficient and robust method to assemble PE molecules or PE-based structures in a particular location and orientation on a Si chip. The method combines PE deposition along a moving contact line onto a poly(dimethylsi­ loxane) (PDMS) ‘stamp’ and microcontact printing (μCP) techniques. Instead of printing of thiol molecular ‘inks’ onto Au films, as in the most common form of μCP, they used stretched and aligned PE strands that act as the ‘ink’ and are printed onto Si chips, with controlled position and orientation (Figure 14). To create patterns of stretched and aligned molecules, a drop of PSA solution was placed with the aid of a pipette onto the micrometer thick PDMS film. Because of the high hydrophobi­ city of the surface, the droplet of the PSA solution only weakly interacts with the surface and remains adhering to the pipette. This fact allows the movement of the droplet in a desired direc­ tion with controlled velocity by moving the pipette. Although visually the droplet does not wet the PDMS surface, a deposition process obviously takes place, which leads to large-area patterns of oriented PSSA fibers (Figure 14). Different structures can be obtained in such a way, such as isolated 1D structures, bundles and rootlike structures depending on deposition conditions. Considering that μCP has been used for wafer-scale pattern fabrication, the method reported here is potentially extendable to a large-scale PE molecules integration and practical produc­ tion of nanodevices and nanosensors.

Chain Conformation and Manipulation

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Contour length (nm) Figure 13 AFM topography images of P2VP (a, b) and poly[methacryloyloxyethyl-dimethyl-(3-pyrrol-1-yl-propyl)ammonium bromide] (PMADAME-Pv) molecules co-deposited with OA by high-speed spin-coating (10000 rpm). Histograms of the contour length distributions for PMADAME-Pv molecules co-deposited with OA by spin-coating at 2000 rpm (a) and 10000 rpm (b). The histograms summarize data for more than 100 molecules from 18 images. From Bocharova, V.; Kiriy, A.; Stamm, M.; et al. Small 2006, 2, 910–916;100 Figure 4 and 5.

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Figure 14 AFM (b–d) and fluorescence (a, e, f) images of PSSA structures formed in different points on the contact line, as shown on (g): in each point the nanostructures are oriented perpendicular to the local moving contact line.

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Another method to manipulate single macromolecules on solid supports is based on transmitting forces across the surface via an ultrathin liquid film. In particular, Severin et al.102 demonstrated that this method may be used to ‘blow’ topolo­ gically or covalently formed polymer loops embedded in an ultrathin liquid film on a solid substrate into circular ‘bubbles’ during AFM imaging. In particular, supercoiled vector ds-DNA has been moved and overstretched to 2 times its B-form length and then torn apart. The blowing of the DNA bubbles can be attributed to the interaction of the tapping AFM tip with the ultrathin liquid film. More specifically, ultrathin liquid films have been prepared by spin-coating of amphiphiles such as octadecylamine onto the basal plane of HOPG. A submono­ layer of a PE such as vector DNA was applied from dilute aqueous solution by putting a drop on the freshly precoated substrate for a few seconds and spinning it off subsequently. Vector DNA molecules were especially suited for such experi­ ments since they are long and monodisperse, and form well-defined polymer rings. The blowing effect was also observed with polymer molecules deposited on the graphite surface coated with an ultrathin solvent film only. Figures 15(a)–15(d) display tapping-mode AFM images of initially supercoiled vector ds-DNA deposited onto graphite covered with butylamine at ambient conditions. A few repeated scans of the whole image caused the two initial loops of the DNA to grow until finally two almost circular bubbles appear. DNA molecules outside the scan area remain in disordered conformations, which was demon­ strated by increasing the scan area after a few scans. In all cases, AFM height and phase images reveal a difference between the inside and the outside of the blowing loops that are identical for all loops in one scan area and does not change during growth of the bubbles (Figures 15(a)–15(d)). Linear polymer molecules that do not form loops are not affected by the scanning. The circumference of the vector ds-DNA circles can become as long as twice the contour length of B-form ds-DNA, indicating that the DNA is overstretched in this state. Large DNA circles can break, and then the height and phase for the area inside quickly level up with the area outside. When the molecular loop is broken, the (a)

force stretching it disappears and the DNA relaxes into a less stretched conformation (Figure 15(f)). After a few scans, the size of the remaining bubbles stabilizes and do not grow anymore under continuous scanning (Figure 15(f)). The DNA loops also grow and can break during repeated tapping in the center of the loops, which was demonstrated by zooming into the center of a DNA loop after the first scan and zooming out after a few scans. This experimental observation implies that the DNA loops can blow in the absence of the direct interaction between the AFM tip and the DNA strand. The transmission of the influence from the tip to the mole­ cule is attributed to the ultrathin liquid film inside the DNA loop. The experimental findings discussed above show that the interaction of the tapping AFM tip with the ultrathin fluid layer creates a new, metastable state of the film inside the loop, characterized by a smaller thickness than the one of the outside film and by a surface pressure, which is higher than the one in the outside layer. This excess pressure causes blowing of the loop into a bubble. When the scanning stops, the metastable state of the film relaxes to a stable one equilibrating its pressure and thickness with those of the outside liquid. Creation of a metastable state is not possible without pumping energy into a system; this energy is provided by the AFM’s tip oscillations.

1.14.8 Nanomanipulations with AFM Tip Common techniques neither allow the control of conforma­ tions at the nanometer scale nor provide a means to obtain specific individual conformations or to control the exact loca­ tion of one macromolecule with respect to another one. On the other hand, the manipulation of single macromolecules on solid substrates with the AFM tip may be used to assemble molecular systems that would not form spontaneously. Considerable progress was made in recent years using the AFM to manipulate nanoscopic objects across surfaces. For a macromolecule, a most critical issue is its interaction with the substrate, which is usually either too weak, causing the

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Figure 15 (a)–(d) AFM tapping-mode height images, showing the unraveling of an initially supercoiled vector ds-DNA on C4H9NH2 upon repeating AFM scanning of the same surface area. (e) During one of the scans, the largest loop breaks and the slow scan direction is indicated by an arrow. (f) After a few scans, the smaller loop stabilizes its size and does not grow under continuous scanning. (a)–(d) from Severin, N.; Zhuang, W.; Ecker, C.; et al. Nano Lett. 2006, 6, 2561–2566;102 Figure 1a–f.

Chain Conformation and Manipulation

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Figure 16 AFM images of ds-DNA adsorbed on a graphite surface modified with CH3(CH2)11NH2 molecules. Manipulation was performed by bringing the tip in contact with the surface and moving it in the desired direction, using homemade manipulation hard- and software. (a) ds-plasmid DNA molecules as deposited. (b) After stretching two of them (nos. 2 and 4) along the white arrows. (c) After manipulation of the same molecules into triangles. From Severin, N.; Barner, J.; Kalachev, A.; Rabe, J.P. Nano Lett. 2004, 4, 577–579;104 Figure 3a–c.

molecule to diffuse rapidly across the surface, or too strong, causing a long molecule to break during manipulation. Kurth et al.103 found that long-chain alkanes and alkylated small mole­ cules self-assemble on crystalline substrates such as the basal plane of graphite into monolayers with the alkyl chains oriented along the substrate axes parallel to each other. Such a ‘molecular carpet’ was found to be an ideal interlayer that reduced interac­ tions between the surface and polymer molecules to an optimal level for nanomanipulations. Figure 16(a) displays the image of four ds-plasmid DNA molecules adsorbed to the modified sub­ strate.104 To subsequently stretch two of the molecules with the AFM, the tip was brought into contact with the substrate and then moved from within a molecular ring outward in four directions as marked in Figure 16(b) to provide a triangular shape. Subsequent imaging in the tapping mode reveals a significant overstretching of the molecules with a factor of about 1.3 relative to their theoretical lengths. The important role of alkylated small molecules preadsorbed onto graphite to form a mobile carpet should be particularly emphasized as no efficient manipulations are possible without such compounds. Dendronized polymers (denpols) were proved to be ideal objects for nanomanipulations and nanoreactions.105 In

particular, Barner et al.106 described the application of a move–connect–prove sequence under ambient conditions to two individual strands of noncharged and charged107 dendro­ nized polymers by using an AFM. The chemical connection between two molecules was achieved by UV irradiation and tested by challenging the mechanical stability of the final mole­ cule. For that purpose, denpols with diameters of a few nanometers and lengths of up to several hundred nanometers were used. They were equipped with peripheral amine groups at every repeat unit, which could be functionalized with azides that easily decompose thermally or photochemically into nitrenes. Nitrenes are highly reactive, short-lived intermediates undergoing various addition and insertion reactions with for­ mation of covalent bonds. Figure 17(b) shows the sample after the two molecules have been moved toward each other with the previously described procedure. After irradiation of the whole sample in situ (while the AFM is scanning) for 3 min with UV-C light, no change of the image was observed (Figure 17(c)). In order to test whether the irradiation caused a stable (covalent) linking of the two adjacent molecules, the mechanical stability of the junction was challenged by pulling on each of the four chain ends with the AFM tip.

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(c)

(d)

(e)

(f)

Figure 17 Tapping-mode AFM images of two individual azide-functionalized dendronized polymers moved toward each other (‘move’; a → b, irradiated by UV light), ‘connect’ (b → c), and challenged mechanically (‘prove’; d–f). The arrows indicate the movement of the AFM tip during manipulation. From Barner, J.; Mallwitz, F.; Shu, L.; et al. Angew. Chem. 2003, 115, 1976–1979;106 Figure 2.

382

Chain Conformation and Manipulation

A critical issue for such move–connect–prove sequences is the preparation of the individual chains on the substrate under conditions in which the adsorption energy has just the right size. On the one hand, it should not be too high, so that the mole­ cules can be moved about with the AFM tip without tearing them apart. On the other hand, it should not be too low, because otherwise the molecules diffuse on the surface until they find each other and form islands to reduce line tension. This subtle balance between opposing factors was achieved by depositing the positively charged denpols on HOPG surface covered by alkylated small molecules. Obviously, the junction did not break (Figure 17(d)). Upon moving through the two points of strongest bending, the molecular chains were cut at the position of impact rather than on the newly formed junction (Figure 17(e)), indicating that the junction is stronger than the main chain.

the composition and constitution of these PE molecules of a complex starlike architecture (Figure 19). The average number of P2VP arms per one molecule was directly estimated from the AFM images.

1.14.10

1D nanostructures of conductive polymers (CPs) have attracted a great deal of interest as building blocks for future miniaturized nanoelectronic devices and highly sensitive chemical112 or bio­ logical sensors.113 Several ‘templateless’ approaches to 1D superstructures of CPs based on the self-assembly of conjugated polymers, oligomers, or monomers have been reported recently.114–116 Other methods for the synthesis of CP nanowires involve chemical or electrochemical oxidative polycondensation in ‘hard templates’ (such as zeolites, track-etched polymeric membranes, and porous alumina),117 or ‘soft templates’ (surfac­ tant micelles or liquid crystalline phases).118 However, for various applications, CP nanowires must be properly integrated into circuits. Therefore, at least one additional step is required, such as the release of the nanowires from the templates and/or their positioning in the device. Polymer chemistry offers a fascinating world of structures of different architecture, composition, and functionality.119 The use of single polymer molecules as templates constitutes a highly promising strategy to generate nano-objects with desired size, shape, location, and specific properties. As demonstrated in previous paragraph, single PE molecules can be stretched and aligned under external forces (such as centrifugal or capillary forces and electric or shear fields) and can be immobilized onto surfaces by simple procedures like casting or printing. Such stretched molecules were used in the preparation of CP nanowires. In the report of Bocharova et al.,120 single chains of PSA were stretched by putting a droplet with PSA solution on a PDMS stamp and then the resulting 1D structures were trans­ ferred from the stamp onto a silicon wafer with gold electrodes.

1.14.9 Chemical Modification of Single Polymer Molecules One of the limitations for the visualization of ‘thin’ PE molecule is the surface roughness of substrates. Topographic contrast in AFM experiments disappears if the substrate roughness is larger than the molecule thickness. This limitation was resolved by the application of either atomically flat substrates (mica, HOPG) or contrasting techniques. The contrasting mechanisms work by the adsorption and electrostatic interaction of charged bulky species (nanoparticles) with the oppositely charged PE molecules. Visualization was drastically improved for posi­ tively charged (in water) P2VP and poly(methacryloyloxyethyl dimethylbenzylammonium chloride) (PMB) molecules by their contrasting with Pd-clusters,108,109 Prussian Blue (PB) clusters, hexacyanoferrate (HCF) counterions (Figure 18),110 and nega­ tively charged Au nanoparticles.111 Contrasting with Pd was applied for the study of a heteroarm star copolymer constituted of seven polystyrene and seven P2VP arms (PS7–P2VP7) on Si wafers and mica substrates.86,87 The AFM visualization revealed

(a)

400 nm before

D

4

3

2

1

L = 2274 nm (b)

400 nm after

D

3

2

1

4

L = 2226 nm 400 nm

after

705 nm 2

1102 nm 1

Nanodevices from Single Polymer Molecules

1105 nm

(c) 419 nm 3 4 411 nm

758 nm

before

Figure 18 AFM topography images of the PMB molecule on Si wafer before (a) and after (b) contrasting with HCF (Z-range 5 nm). The AFM data indicate that increase of the chain thickness from a few angstroms to 1.5 nm upon treatment with K4Fe(CN)6 occurs virtually without any changes in their location, contour length, and fine details of the conformation within the resolution limit. Snapshot (c) demonstrates the transition of the 2–3 segment induced by the contrasting procedure. From Kiriy, A.; Gorodyska, G.; Minko, S.; et al. J. Am. Chem. Soc. 2003, 125, 11202–11203;110 Figure 2.

Chain Conformation and Manipulation

383

(a)

100 nm 9

Height (nm)

(c) (b) 50 nm

6 3 0

0

200

400 nm 7 nm

(d) 100 nm

Figure 19 3D (a, b) and 2D (c) AFM images, cross section (d), and schematic representation (d) of Pd-metallized linear (a) and star-shaped PS7–P2VP7 (b–d) molecules. From Kiriy, A.; Gorodyska, A.; Minko, S.; et al. Macromolecules 2003, 36, 8704–8711;87 Figure 3.

AFM investigations reveal that PSA molecules form bridges between gold electrodes. To produce polypyrrole (PPy) nano­ wires, a kind of ‘electroless’ deposition of PPy was performed. For that, the chips with deposited PSA bridges were incubated with aqueous solution of pyrrole (Py) and APS. The diameter of the grown nanowires can be varied from a few nanometers to hundreds of nanometers by adjusting the polymerization time and concentration of the reagents. Electrical characterization was performed for multiple PPy bridges as well as for single PPy nanowires (Figure 20). For investigations of electrical properties of single PPy nanowires, the amount of (50 nm thick) PSA–PPy

nanowires produced was adjusted to allow only a few Ppy nanowires to bridge the gold microelectrodes (the distance between the electrodes was 1 μm). The nanowires display a linear current–voltage dependence (Figure 20(f), inset), which reflects good contacts between the nanowires and the electrodes and good connectivity of the PPy clusters along the PSA molecule. The dc-conductivity of individual PPy nanowires approached the conductivity of PPy in bulk (0.1 and 3 S cm−1). To break a certain nanowire, we slowly increased the potential between the electrodes until an abrupt increase in resistance was observed. The significant resistance increase after this procedure

(a)

(f) R ~ 21 MΩ 20

0.2

(b)

400 nm

(c)

I (μA)

R (MΩ)

15 1000 nm

10

0 –0.2 –0.2 0

5 (d)

0.2

U (V) R ~ 1.1 MΩ

(e) 0 –100

–50

0

50

100

U (V)

Figure 20 AFM topography images of the representative Ppy nanowire with the resistivity of about 1 MΩ (a); high-magnification topography AFM images (b–c) of the pristine Ppy nanowire bridged between two microelectrodes (b, d); the same nanowire was broken applying high voltage (25 V) (c, e). (h) Room-temperature resistivity–current characteristics of the pristine nanowire (red line); the same nanowire after the breaking with high voltage (green line); inset in (f) demonstrates the linear I–V dependence for the pristine nanowire at low voltage (blue line). From Bocharova, V.; Kiriy, A. Vinzelberg, H.; et al. Angew. Chem. 2005, 117, 6549–6552;120 Figure 2.

384

Chain Conformation and Manipulation

(from 1.1 to 21 MΩ for the nanowire in Figure 20(b)) proves that the measured conductance is indeed caused by this particu­ lar nanowire. On the other hand, AFM inspection of the broken nanowires reveals the formation of gaps (usually one or two per broken wire; Figure 20(b)–20(e)). An even more pronounced decrease of the conductance (the resistance increased from a few megaohms to a few gigaohms) was observed when the nano­ wires were mechanically broken by movement of the AFM tip (operating in contact mode) across the wire. The possibility of using PPy nanowires as active elements in sensors was demonstrated. In general, the conductivity of PPy can be modulated by changing the doping level: the conductiv­ ity is high in an oxidized (doped) state and low in a reduced (de-doped) form. Alternatively, PPy in the reduced form can be doped by acids and dedoped by bases. To make the conductiv­ ity of the PPy nanowires sensitive to acids and bases, they were reduced (dedoped) by rinsing with an aqueous solution of NaBH4 (1 g l−1), which resulted in an increase in resistance from 1 MΩ for doped nanowires to more than 1 GΩ for the dedoped wires. In the reduced state, the nanowires reversibly changed their resistance upon exposure to HCl and NH3 vapors from several megaohms to a few gigaohms.

1.14.11

Conclusions and Outlook

Individual polymer molecules can be visualized, manipu­ lated, and to some extent controlled on solid substrates. This fascinating area of polymer research has become possible due to the progress with scanning microscopic techniques. It first allows polymer conformations under different adsorption conditions to be analyzed and in this way to obtain further information about 2D and also 3D conformations prior to the adsorption step. Here the adsorption process itself, but also equilibration on the surface, might play a role. It was, how­ ever, possible to get insight into particular PE conformations and phase transitions, for example. Second, the molecules on the surface can even be monitored in situ or in steps to obtain information on molecular motion on the surface. This requires the control of surface interaction forces for a delicate balance of adsorption strength versus mobility of the chains on the surface. There are also first attempts to obtain con­ trolled and directional motion of molecules on the surface. Third, the single molecules have been shown to act as tem­ plates for deposition of nanoparticles and can be used for nanodevices and sensors. Single polymer molecules will be further analyzed and manipulated on surfaces. With advancing techniques, one can expect further improvement of resolution and scanning modes. So different properties including electron distribution, atomic vibrations, and local dynamics might become accessible. On the other hand, various manipulation tools might be developed to allow further manipulation of conformations and chemistry at molecular level. This could lead to many new possibilities of applications at molecular and nanoscopic level. One of the main problems for widespread applications is, however, still the limitation to single cantilever AFM of each manipulation being time-consuming and tedious. Possibly, self-assembly pro­ cesses can help, and with synthetic polymer molecules one could use the overwhelming variety of existing monomers and

molecular architectures. So we believe that single-molecule poly­ mer physics and chemistry still has a bright future.

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Biographical Sketches Anton Kiriy is a senior scientist in the Department of Nanostructured Materials at Leibniz Institute for Polymer Research (IPF) in Dresden, Germany. Anton Kiriy received his diploma degree in 1988 in organic chemistry at the University of Kiev (KPI, Ukraine). He defended his Ph.D. degree in organic chemistry in 2000 in the same university. Since 2001, he has been a staff scientist at Leibniz Institute of Polymer Research Dresden, Germany. His research interests range from single molecule studies and molecule-based functional nanodevices, to controlled preparation of conjugated polymers, synthesis and applications of nanostructured conjugated polymers.

Since 1999, Manfred Stamm has been Professor of Physical Chemistry of Polymeric Materials at Technische Universität Dresden and Head of the IPF Institute of Physical Chemistry and Physics of Polymers at Leibniz Institute of Polymer Research Dresden, Germany. From 1979 to 1985, he was a staff scientist at the Institute of Solid State Research in Jülich. He did his research on small-angle neutron scattering, chain conformation, preparation of polymers, and conductive polymers. From 1984 to 1985, he was a visiting scientist at Brookhaven National Laboratory in the United States (neutron reflectometry, polymer interface investigations, and interdiffusion studies). From 1985 to 1999, he was a staff scientist and project leader at Max-Planck Institute of Polymer Research in Mainz, Germany (interfaces between polymers, structure and conformation, phase transitions, and development of scattering techniques). He received the International Belgien Polymer Group Award in 2004.