Nanoscale Memory Provided by Thermoreversible Stochastically

Departamento de Quı´mica Orga´nica, UniVersidad de Buenos Aires, C1428EHA ... Argentina, and Department of Chemistry, Purdue UniVersity, West Lafay...
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Langmuir 2006, 22, 9682-9686

Nanoscale Memory Provided by Thermoreversible Stochastically Structured Polymer Aggregates on Mica Avishay Pelah,*,† Silvio J. Luduen˜a,‡ Elizabeth A. Jares-Erijman,§ Igal Szleifer,£ Lı´a I. Pietrasanta,‡ and Thomas M. Jovin*,† Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, D-37070 Go¨ttingen, Germany, Centro de Microscopı´as AVanzadas, Facultad de Ciencias Exactas y Naturales, and Departamento de Quı´mica Orga´ nica, UniVersidad de Buenos Aires, C1428EHA Buenos Aires, Argentina, and Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907 ReceiVed December 19, 2005. In Final Form: May 11, 2006 Stimuli-responsive polymers are used in a large variety of applications due to the controlled manner in which their physical properties can be reversibly altered. In this study, we demonstrate the thermoreversible structuring of poly(N-isopropylacrylamide)-based polymer. By temperature-controlled atomic force microscopy, we demonstrate that polymer aggregates form on mica above the polymer lower critical solution temperature and disperse below it, and in so doing, display positional “memory” in that the nanodomains are retained in the same positions and with the same shapes during repeated cooling/heating cycles. Such positional “memory” may be useful for multiple applications in nano-microscale devices.

Introduction Stimuli-responsive polymers, also known as “smart” polymers, are of great utility in fields such as biomedicine and nanotechnology. These materials exhibit substantial changes in their properties in response to relatively minor alterations in the microenvironment. The different stimuli inducing such transitions include shifts in temperature, ionic strength, and pH. Poly(Nisopropylacrylamide), PNIPAM, is a water-soluble polymer with a lower critical solution temperature (LCST) of 32 °C.1 Heating an aqueous solution above the LCST leads to dehydration of the polymer chains by expelling water, resulting in a thermoreversible coil-to-globule phase transition and chain aggregation. Due to their unique thermoresponsive properties, polymers based on N-isopropylacrylamide have been at the focus of numerous studies 2-10 and applications such as bioanalytical assays,3,6,11 manipulation of aggregate size,4 and the control of enzymatic activity.7 * To whom correspondence should be addressed. E-mails: [email protected] (A.P.); [email protected] (T.M.J.). † Max Planck Institute for Biophysical Chemistry. ‡ Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. § Departamento de Quı´mica Orga ´ nica, Universidad de Buenos Aires. £ Purdue University. (1) Schild, H. G. Poly(N-isopropylacrylamide): experiment, theory and application. Prog. Polym. Sci. 1992, 17, 163-249. (2) Bergbreiter, D. E.; Caraway, J. W. Thermoreponsive Polymer-Bound Substrates. J. Am. chem. Soc. 1996, 118, 6092-6093. (3) Hoffman, A. S. Bioconjugates of Intelligent Polymers and Recognition Proteins for Use in Diagnostics and Affinity Separations. Clin. Chem. 2000, 46, 1478-1486. (4) Kulkarni, S.; Schilli, C.; Mu¨ller, A. H. E.; Hoffman, A. S.; Stayton, P. S. Reversible meso-scale smart polymer-protein particles of controlled sizes. Bioconjugate Chem. 2004, 15, 747-753. (5) Li, C.; Gunari, N.; Fischer, K.; Janshoff, A.; Schmidt, M. New Perspectives for the Design of Molecular Actuators: Thermally Induced Collapse of Single Macromolecules from Cylindrical Brushes to Spheres. Angew. Chem., Int. Ed. 2004, 43, 1101-1104. (6) Malmstadt, N.; Hyre, D. E.; Ding, Z.; Hoffman, A. S.; Stayton, P. S. Affinity Thermoprecipitation and Recovery of Biotinylated Biomolecules via a Mutant Streptavidin-Smart Polymer Conjugate. Bioconjugate Chem. 2003, 14, 575-580. (7) Shimoboji, T.; Larenas, E.; Fowler, T.; Hoffman, A. S.; Stayton, P. S. Temperature-Induced Switching of Enzyme Activity with Smart Polymer-Enzyme Conjugates. Bioconjugate Chem. 2003, 14, 517-525. (8) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G.; Harris, J. M.; Hoffman, A. S. Control of protein-ligand recognition using a stimuliresponsive polymer. Nature 1995, 378, 472-474.

Aqueous solutions of PNIPAM phase-separate so as to form colloidally stable particles when the temperature is raised above the LCST.12 These particles are stabilized by electrostatic repulsion between sulfate groups originating from the persulfate initiator.12 In the present study, carried out by temperaturecontrolled tapping mode atomic force microscopy (AFM) in water, we demonstrate a new and intriguing “memory” property of PNIPAM-based polymer aggregates formed on a mica surface, namely the memorization of their positions and shapes over numerous cooling and heating cycles despite the loss of apparent structure at the lower temperature. These structures evolve in the course of heating via the incorporation of polymer from the overlying solution and by the coalescence of neighboring aggregates. The phenomenon has been characterized and rationalized by quantitative image analysis and has numerous practical implications. Materials and Methods Polymer Synthesis. The PNIPAM polymer was synthesized according to Bokias et al.13 We incorporated during synthesis a 2.5% weight fraction of an amine-bearing monomer, N-(3-aminopropyl)methyacrylamide (Polysciences). The polymer had an Mn of 46 kDa and an Mw of 61 kDa, according to gel permeation chromatography, and an Rh of 4.8 nm, determined by dynamic light scattering (GPC and DLS were performed at the Max Planck Institute for Polymer Research, Mainz, Germany). We further reacted the polymer in a 0.1 M sodium bicarbonate buffer (pH 8) with EZ-link Sulfo-NHS-LC-Biotin (Pierce). A incorporation of ∼3.3 biotins per (9) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, T. Temperature-Responsive Bioconjugates. 1. Synthesis of Temperature-Responsive Oligomers with Reactive End Groups and Their Coupling to Biomoleculs. Bioconjugate Chem. 1993, 4, 42-46. (10) Zhu, M.; Wang, L.; Exarhos, G. J.; Li, A. D. Q. Thermosensitive Gold Nanoparticles. J. Am. Chem. Soc. 2004, 126, 2656-2657. (11) Ding, Z.; Chen, G.; Hoffman, A. S. Synthesis and Purification of Thermally Sensitive Oligomer-Enzyme Conjugates of Poly(N-isopropylacrylamide)-Trypsin. Bioconjugate Chem. 1996, 7, 121-125. (12) Chan, K.; Pelton, R.; Zhang, J. On the Formation of Colloidally Dispersed Phase-Separated Poly(N-isopropylacrylamide). Langmuir 1999, 15, 4018-4020. (13) Bokias, G.; Durand, A.; Hourdet, D. Molar mass control of poly(Nisopropylacrylamide) and poly(acrylic acid) in aqueous polymerizations initiated by redox initiators based on persulfates. Macromol. Chem. Phys. 1998, 199, 1387-1392.

10.1021/la053431+ CCC: $33.50 © 2006 American Chemical Society Published on Web 09/19/2006

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chain was achieved according to a HABA/Avidin assay.14 The number of remaining amino monomers per polymer chain was determined by a fluorescamine assay,15 with the amino monomer as control. A temperature-controlled Varian Cary 100 spectrophotometer was used for the determination of the polymer LCST. The LCST, defined as the midpoint of the thermal transition measured by the absorbance at 600 nm of a 2 mg/mL aqueous polymer solution, was 32.4 °C. AFM Experiments. Samples were scanned in a fluid cell of a Digital Instruments MultiMode scanning probe microscope IIIa (Veeco). The liquid chamber of the AFM was filled with a 0.5-5 µM salt-free aqueous solution of the polymer in contact with a freshly cleaved mica surface [G 250-1, KAl2(OH, F)2(AlSi3O10), Plano], and the latter was scanned at temperatures below and above the LCST. The temperature was controlled by a home-built, two-stage Peltier temperature controller, which achieves high positional stability by maintaining the scanning head at constant temperature, while adjusting the temperature of the polymer solution as desired. Scans (256 × 256; 512 × 512) were always from “top to bottom” at 1-8 Hz line frequency. Image Analysis. Images were flattened with the NanoScope software and were further analyzed with DIPImage (TNO, Delft University of Technology). Background was subtracted from each image. An aggregate was defined as a cluster of at least 50 pixels with a mean height of at least 1 nm. The circularity of an object in 2D is defined as the square of the perimeter divided by 4π‚surface area covered by the object; the minimal value of 1 corresponds to a circle.

Results and Discussion The use of a temperature-controlled AFM allows the nanoscale investigation of PNIPAM aggregates, as well as other temperatureresponsive materials such as PNIPAM hydrogels, below and above their LCST.16 Our AFM experiments were first aimed at the characterization of biotinylated PNIPAM aggregates. The incorporation of the hydrophilic amine comonomer to the PNIPAM polymer increased the LCST 17 to ∼40 °C, which was again lowered to 32.4 °C upon enhancement of the hydrophobic character of the polymer by the incorporation of biotin. Biotinylation was incomplete, such that ∼ 2.3 free amino groups per polymer chain were retained. The protonated amino groups presumably facilitated the adsorption of the polymers to the negatively charged mica surface. The phenomena described below did not occur in PNIPAM polymers lacking amines. Figure 1A (top, left column, images of cycle 2) depicts the appearance and growth of polymer aggregates upon raising the temperature from 30 to 38 °C. After the system stabilized (Figure 1A, second row, left column), the temperature was lowered below the LCST, resulting in the complete dissolution of the aggregates such that no remaining structures were perceived (Figure 1A, bottom left image). The process of aggregation/dissolution reoccurred during the course of further heating/cooling cycles, with the unexpected finding that the aggregates reformed with the same geometric distributions characteristic for each temperature as exhibited in the first cycle (Figure 1A, right column images of cycle 4). Varying the polymer concentration led to systematic changes in the number, size, and shape of the polymer aggregates. The water solubility and small dimensions of individual polymer molecules (Rh ) 4.8 nm) accounted for the inability to (14) Green, N. M. A spectrophotometric assay for avidin and biotin based on binding dyes by avidin. Biochem. J. 1965, 94, 23-24c. (15) Bohlen, P.; Stein, S.; Dairman, W.; Udenfriend, S. Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 1973, 155, 213-220. (16) Matzelle, T. R.; Geuskens, G.; Kruse, N. Elastic Properties of Poly(Nisopropylacrylamide) and Poly(acrylamide) Hydrogels Studied by Scanning Force Microscopy. Macromolecules 2003, 36, 2926-2931. (17) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Effect of Comonomer Hydrophilicity and Ionization on the Lower Critical Solution Temperature of N-Isopropylacrylamide Copolymers. Macromolecules 1993, 26, 2496-2500.

Figure 1. “Memory” of PNIPAM-based polymer aggregates on mica revealed by temperature-controlled AFM. (A) Surface plots of the reversible aggregation of biotinylated amino-PNIPAM on mica upon heating-cooling cycles between 30 and 38 °C. Cycles 2 and 4 are depicted. Row 2: aggregates maintained at 38 °C. Mean aggregate height at 38 °C: cycle 2, 3.8 nm; cycle 4, 4.2 nm. Rows 3 and 4: transition and maintenance of an unstructured, dissolved state below the LCST. Scans: top to bottom; frame rate 8 Hz; field 3 × 3 µm2, 256 × 256 pixels; surface view: rotation 0°, pitch 75°. Polymer concentration, 3 µM. (B) Top AFM view of a polymer layer remaining on mica after a short exposure to 1 µM of polymer solution below the LCST. The mica was thoroughly washed several times with cold water, dried, and scanned. Scan size: 1 × 1 µm2, 512 × 512 pixels.

detect them on the mica surface under liquid and below the LCST. However, measurements by ellipsometry in solution confirmed the existence of a thin polymer layer on the mica surface at temperatures below the LCST (data not shown). Furthermore, AFM in air resolved polymer molecules on a mica surface exposed for a short time to the polymer solution, washed thoroughly in water, and dried in air (Figure 1B). Due to technical difficulties involved in switching between imaging in air and liquid, we could not acquire images in liquid from the identical region scanned in Figure 1B. Nevertheless, we conclude that polymer chains adsorb tightly to the negatively charged mica surface at low temperatures by exchange processes and electrostatic interactions involving the amine moieties and serve as nucleation sites for subsequent aggregation above the LCST. Although appearing densely packed in air, the features shown in Figure 1B may constitute the templates for the micrometerscale “memory” effect due to the heterogeneity of the polymer charge (different number of contact points per chain) and the mobility of its aggregates above the LCST, as discussed below. The memory effect was abolished by exposure to salt solutions, which led to detachment of the polymer from the surface due to the presence of competing cations and anions. Similar experiments performed on highly ordered pyrolytic graphite (HOPG) did not result in the formation of discreet structures but rather led to a complete coverage of the surface by the polymer above the LCST and the dissolution of the layer below the LCST (not shown). The adsorption results from the high affinity of the

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Figure 2. Formation of “memory aggregates”. (A) Sequential scanning of the same area during three heating/cooling cycles (see also Movie 1S in the Supporting Information). Scans were top to bottom at 4 Hz (scan size 3 × 3 µm2, 512 × 512 pixels), and the polymer concentration was 2 µM. Scans were taken 2 min after each stepwise shift in temperature. (B) Overlap images of the three 40 °C scans. Left to right: overlap (yellow): 5, 14; 14, 21; 5, 21.

hydrophobic HOPG for the hydrophobic polymer above the transition temperature. This adsorption was insensitive to polymer charge. Figure 2A (see Movie 1S in the Supporting Information) depicts a series of scans of a given area during three heating/cooling cycles. The formation, dissolution, and reformation of polymer aggregates was highly reproducible, as demonstrated by the >60% overlap of the binary masks at 40 °C (Figure 2B). No apparent structures were detectable below a threshold temperature near the LCST. However, they (re)appeared as a pattern of small domains that evolved to the final aggregates as the temperature was systematically increased. Various features and parameters characterizing this process were derived by quantitative image processing of the AFM data (Figure 3). The number of aggregates resolved by the AFM increased up to the LCST but decreased sharply at higher temperatures (Figure 3), owing to the coalescence into larger structures. An accumulation of polymer from the overlying solution onto the growing aggregates upon heating could be deduced from the mean height, area, and volume of the aggregates, as well as from the total aggregate volume per image field (Figure 3). [A peak in the number of aggregates at the lowest temperature,

Figure 3. Parameters for PNIPAM domains derived from image analysis: number of aggregates per image field; mean aggregate area, height, volume, and circularity; and total volume of aggregates per image field. The numbered bars correspond to the image numbers of Figure 2A. Vertical error bars, standard deviations. See materials and methods for image analysis information.

at which the volume, height, and area parameters are minimal, indicates the existence of minute structures (approximately the

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Figure 4. Coalescence of neighboring aggregates. Surface plot images depicting dynamic coalescence (5-9) and its reversibility (1-4) in biotinylated amino-PNIPAM aggregates formed from a 2 µM solution at 38 °C (see also Movie 2S in the Supporting Information). Mean aggregate height 5.5 nm. Scan details: direction top to bottom: 8 Hz; original scan size 1.5 × 1.5 µm2, 256 × 256 pixels, shown with a 2-fold zoom; rotation 0°, pitch 80°.

size of our default definition of an aggregate; see Materials and Methods) that has been resolved by the AFM.] All of these parameters peaked at the highest temperature, although there were interesting deviations from symmetrical behavior about this point (compare images at corresponding temperatures in Figure 2A). Hysteresis was observed in the mean aggregate area and volume but not the height, such that the values at a given temperature on the ascending limb were considerably smaller than those at the corresponding temperature on the descending limb of the transition [see Figure 2A and Figure 3: 34 °C, [2,12,19 ] < 7 (area ratio 2.0-2.6; volume ratio 3-7); 35 °C, [3,13] < 6 (area ratio 1.6; volume ratio 2)]. In contrast, the number of aggregates showed the inverse relationship, i.e., was lower on the descending limb. Inasmuch as the total volume remained constant, we infer that the hysteresis reflected the maintenance of the state of maximal coalescence achieved at the highest temperature during the cooling phase. Interestingly, in the first heating-cooling cycle, the mean aggregate area at 40 °C was less than at the straddling temperatures of 37 and 35 °C. This feature arose from the greater degree of compaction at the highest temperature but was not resolved in subsequent cycles because of the larger temperature steps employed. The decrease at higher temperatures in mean circularity (a parameter that assumes the minimal value of 1 for a circle but is >1 for any other shape;17 Figure 3) provided an additional measure of the compaction achieved by solvent exclusion and coalescence, acting to minimize surface contact between polymer and solvent. Upon prolonged incubation, the mobility of “mature” hightemperature aggregates was manifested by further alterations in size and shape, leading to the coalescence (coagulation) of initially separated domains after brief contact. To visualize this process, we continuously scanned a polymer-coated mica surface at 38 °C and at a high scan rate (8 Hz line frequency, corresponding to ∼30 s per image). Figure 4 (1-3 and 5-9) depicts two coalescence events (see Movie 2S in the Supporting Information). This feature was hierarchical, as if the propensity of one particle to join (“consume” or “be consumed by”) another depended upon the relative strengths of the respective anchors of an aggregate to its nucleation site. In the case of tightly bound aggregates, the coalescence with an adjacent partner was also reversible (Figure 4 (1-4)). The driving force underlying the process featured above is presumably the energetically favorable minimization of contact surface of the polymer with the solvent operating through two different mechanisms. According to the first, polymers adsorbed

Figure 5. Proposed mechanism for “polymer memory”. (A) Polymer solution is added to the mica below the LCST. (B) Polymer chains (black), bearing different numbers of amine moieties, attach to the mica and form a heterogeneous layer. More chains (gray) remain in an extended form in solution. (C) As the temperature is raised above the LCST, lateral collapse of polymer attached to the mica creates nucleation sites and collapsed polymer chains and aggregates from solution adsorb to these domains (and to some extent outside them), causing the lateral (including coalescence) and vertical growth of the aggregates. Red particles represent polymers that attach to mica regions exposed upon lateral collapse of the initial layer, thereby increasing the local density. (D) (1) Neighboring aggregates grow and coalesce to form larger particles. (2) Upon cooling below the LCST, the aggregates dissolve, leaving behind a nucleation “template”. (3) Reheating above the LCST induces the formation of aggregates at the same positions dictated by the template. (1), (2), and (3) repeat during further heating/cooling cycles. See text for further details.

onto other molecules but lacking direct electrostatic interaction with the mica surface via their charged amines, act as bridges between neighboring aggregates and thereby facilitate their rearrangement into preferential larger domains. [The height measurements (Figure 3) indicate that the aggregates comprised more than one molecular layer.] The second mechanism entails bound polymer serving as “rails” facilitating the translocation of mobile aggregates into larger structures. Although we cannot rule out an additional dragging effect exerted by the AFM tip, coalescence events were observed at quite different times and in the absence of continuous scanning. Furthermore, the scans were reproducible, excluding tip effects as the primary factor. Zhao et al. 18 used AFM to demonstrate the “clumping” of an end-grafted poly(styrene) on silicon into islands as the grafting density decreased. On the basis of AFM force measurements, Kelley et al.19 discerned regions of high and low polymer density of end-grafted poly(2-vinylpyridine)polystyrene on mica. These observations of heterogeneity in the density of a polymer grafted to surfaces (polymer brush) in a poor solvent environment and the particular nature of the polymer that we studied lead us to propose the following mechanism for “PNIPAM memory” (Figure 5). The amine-bearing PNIPAM-based polymers adsorb stochastically to the negatively charged mica surface below the LCST, forming domains of higher and lower chain density. (18) Zhao, W.; Krausch, G.; Rafailovich, M. H.; Sokolov, J. Lateral Structure of a Grafted Polymer Layer in a Poor Solvent. Macromolecules 1994, 27, 29332935. (19) Kelley, T. W.; Schorr, P. A.; Johnson, K. D.; Tirrell, M.; Frisbie, C. D. Direct Force Measurements at Polymer Brush Surfaces by Atomic Force Microscopy. Macromolecules 1998, 31, 4297-4300.

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polymer adsorbs onto the nucleation sites causing smaller aggregates to coalesce, thus forming fewer and larger structures deviating from the more spherical smaller aggregates. This description of molecular events at the liquid-solid interface does not exclude the intervention of direct absorption from solution of preformed particles during the first thermal cycle, which may account for a fraction of the stochastic pattern of discrete domains.

Summary

Figure 6. Pattern formation. (A) Sequential scanning of a cross pattern formed by scanning in contact mode below the LCST, followed by heating. Shown is a subsequent cooling and heating cycle (see also Movie 3S in the Supporting Information). Polymer concentration, 5 µM. Scans: top to bottom; 8 Hz; scan size 10 × 10 µm2, 512 × 512 pixels. (B) Overlap of the two patterns formed at the high temperature (40 °C) demonstrating the “memory” effect (overlap in yellow).

Variations in density also arise from inherent polymer heterogeneity, reflected as the differential adsorption of mono- and multi-amine-bearing polymer chains. The latter tend to engage in multipoint contacts spanning a surface comparable to that occupied by several mono-amine chains attached at an end or point but otherwise extending into solution. Furthermore, fluctuations in the local structure and physicochemical properties of the mica surface20 may define preferential loci for adsorption. Raising the temperature above the LCST sharply reduces the solvency of water for the polymer, resulting in the lateral collapse of adjacent extended polymer chains, which fuse in order to maximize inter- and intrachain interactions and minimize chainsolvent interaction, creating minute condensed islands. As aggregation is hydrophobically driven, such domains function as nucleation sites for the incorporation of polymer from the overlying solution, thereby promoting both lateral and vertical growth and, ultimately, nearest-neighbor coalescence. Lateral collapse of polymer chains on the surface exposes unoccupied adsorption sites for additional amine-bearing polymer chains and particles, leading to a further increase in the local density about the template. The slight increases during successive cycles of the mean aggregate height and volume values at 40 °C (Figure 3) implies a progressive consolidation of the overall process. Upon cooling, the aggregates dissolve and the individual chains return to solution although the nucleation domains fixed to the mica remain and serve again as templates in subsequent thermal cycles. According to the proposed mechanism, the number, size, and shape of aggregates would be dependent upon the concentration of the polymer in solution. At higher concentrations, more (20) Jiao, Y.; Cherny, D. I.; Heim, G.; Jovin, T. M.; Scha¨ffer, T. E. Dynamic Interactions of p53 with DNA in Solution by Time-Lapse Atomic Force Microscopy. J. Mol. Biol. 2001, 314, 233-243.

PNIPAM-based polymers are versatile, and their properties can be tailored to meet specific needs. Their utility for surface patterning and nanoparticle organization was recently reported.21 The tapping-mode AFM experiments in water described here have revealed “nanodomain memory”, a novel and potentially very useful property of thermoresponsive PNIPAM modified by random insertion of amine groups. The physical origin of the memory effect arises from the seemingly irreversible adsorption of a few molecules to the mica surface. Changes in the quality of the solvent forces a constrained collapse and expansion of the PNIPAM chains due to the confinement imposed by the adsorbed polymer chains, which serve as reproducible templates over repeated cycles of heating and cooling. The determination of the position and specificity of the aggregates depend on the chemical composition and sequence of the polymers and the detailed local structure of the mica surface. Tuning the polymer by incorporation of particular hydrophilic and/or hydrophobic moieties provides the means for controlling important features of the resultant aggregates. These features can be deterministic, e.g., achieved by mechanical manipulation of the mica (or other) surface, demonstrated by the simple cross pattern in Figure 6A and B (generated by pressing on the mica surface with the AFM tip in contact mode; see Movie 3S in the Supporting Information). It is also possible to clear rectangular areas by sweeping with the AFM tip and thereafter re-establish new and different aggregation patterns within these regions (we will report elsewhere on these phenomena). The introduction of ordered and chemically distinctive nucleation sites may lead to novel applications in the booming field of nanotechnology, as well as in larger scale systems. For example, one can envision arrays differing in affinity for the PNIPAM polymers and/or alternative stimuli-sensitive materials and exploiting the coalescence and other properties featured in this report. Acknowledgment. Thanks are due to Dr. Rio Kita and Christine Rosenauer from the Max Planck Institute for Polymer Research in Mainz for the physical characterizations of the polymers, as well as to Gudrun Heim and Dr. Keith Lidke for assistance with the AFM measurements and image analysis, respectively. We thank Dr. Stefan Ho¨ppner for help with the illustrations. I.S. acknowledges the National Science Foundation (CTS-0338377) for partial support of this work. L.I.P. is a permanent research fellow of CONICET (Argentina). Supporting Information Available: Movies 1s, 2s, and 3s, depicting sequence of events shown in Figure 2A, Figure 4, and Figure 6, respectively. This material is available free of charge via the Internet at http://pubs.acs.org. LA053431+ (21) Lee, L. T.; Leite, C. A. P.; Galembeck, F. Controlled Nanoparticle Assembly by Dewetting of Charged Polymer Solutions. Langmuir 2004, 20, 4430-4435.