Chemical Contrasting in a Single Polymer Molecule AFM Experiment Anton Kiriy,* ,† Ganna Gorodyska, † Sergiy Minko,* ,†,‡ Constantinos Tsitsilianis, § Werner Jaeger, | and Manfred Stamm † Institut fu ¨r Polymerforschung Dresden, Hohe Strasse 6, 01069 Dresden, Germany, Department of Chemistry, Clarkson UniVersity, Potsdam, New York 13699-5810, Department of Chemical Engineering, UniVersity of Patras, 26504 Patras, Greece, and Fraunhofer-Institut fu ¨r Angewandte Polymerforschung, Geiselbergstrasse 69, 14476 Golm, Germany Received July 14, 2003; E-mail: sminko@clarkson.edu; kiriy@ipfdd.de Atomic force microscopy (AFM) is used for the visualization of single polymer molecules and the probing of their properties. 1 New knowledge was obtained from the AFM study of polymer adsorption and reconformation in a controlled environment, 2 esti- mation of molecular weight, 3 and monitoring of chemical reactions between single polymer molecules. 4 The experiments were per- formed mainly with relatively “thick” polymeric molecules if the diameter of the chain was larger then 1 nm: dendronized polymers, 4 “molecular brushes”, and some naturally occurring polymers. 1 How- ever, most of the synthetic polymers and many natural polymers have much thinner chains. 5 Although several methods to improve the detection of polymer chains via chemical modification of tips or applying special AFM modes have been proposed, 1f,g the de- velopment of techniques for the study of the conformation of iso- lated chains with molecular resolution on rough surfaces is still a challenging task. Recently, we succeeded in visualizing polycations (PC) in different conformations adsorbed onto the atomically flat mica. 2 Although these PC are invisible on a Si-wafer due to the high roughness of the substrate, 6 they can be resolved after the decoration of the deposited chains with Pd clusters. 7 Metallization of PC causes the strong contraction of chains (2-3 times decrease of the contour length) even if the chains were strongly trapped by the substrate. The local collapse of the chain was induced by interaction with the bivalent ion PdCl 4 2- . Here, we report for the first time on the contrasting method for AFM to substantially improve the resolution of a single molecule experiment with no changes of the conformation of adsorbed poly- mer molecules. In our approach, we use the deposition of either hexacyanoferrate (HCF) anions or negatively charged clusters of cyanide-bridged complexes as contrasting agents (Figure 1a). This method allowed us to increase the thickness of the resulting struc- tures up to 3 nm and, consequently, to provide visualization of poly- mer chains on Si-wafers. After AFM-measurements, the contrasting agents were then removed also without distortion of the molecule conformation. We used in this study three different PC molecules: poly(meth- acryloyloxyethyl dimethylbenzylammonium chloride) (PMB) 2a M W ) 6130 kg/mol, poly(2-vinylpyridine) (P2VP) 2b M W ) 385 kg/ mol, and the star-shaped heteroarm block copolymer with seven polystyrene M W ) 20 kg/mol and seven P2VP M W ) 56.5 kg/mol arms (PS 7 -P2VP 7 ). 2c Both PMB and P2VP molecules deposited onto the Si-wafer are not resolved in the tapping mode (Figure 1b). 8 Although PS 7 -P2VP 7 molecules adsorbed onto mica from acid water (pH 2) solution display a clear core-shell morphology (Figure 1e), 2c only the core of unimers with the height of 5 nm can be resolved on the Si-wafer (Figure 1f). We were successful in visualizing all of these polymers on the Si-wafer substrate with the following staining procedure. The samples were prepared as described else- where. 2,7 The polymer molecules were deposited on the freshly cleaved mica or Si-wafers from a very diluted (0.0005 mg/mL) acid solution (pH 2.5-3). The drop of the examining solution was set on the substrate for 60 s, and afterward it was removed with a centrifugal force. The molecules were examined in a dry state with a Multimode AFM instrument (Digital Instruments, Santa Barbara) in the “light” tapping mode. Details of the AFM experiments were reported elsewhere. 2,7 The samples were then stained upon exposure for 3 min to an HCF acid solution bath. The sample was rinsed in water and dried for the AFM experiment. Figure 1c,g presents AFM images of PMB and PS 7 -P2VP 7 molecules contrasted with HCF. In all cases, we observed an 0.6-0.7 nm increase of heights of resulting structures, 9 that roughly corresponded to the size of the HCF-anion (Figure 1a). We found a strong effect of pH on the contrasting process. No attachment of HCF-anions was observed at pH higher than 4, that is, above the isoelectric point of the Si- † Institut fu ¨r Polymerforschung Dresden. ‡ Clarkson University. § University of Patras. | Fraunhofer-Institut fu ¨r Angewandte Polymerforschung. Figure 1. Scheme of the contrasting of adsorbed polycations (a), AFM topography images (b,c), and a cross-section (d) of PMB, and AFM images of PS7-P2VP (e-g) molecules before (b,e,f) and after (c,g) contrasting with HCF (Z-range 5 nm). All images are on Si-wafers, but (e) is on mica. Histograms of molecular diameter distribution for PS 7-P2VP7 adsorbed onto the mica, with no contrasting, are shown in (h), and those onto the Si- wafer after contrasting with HCF are shown in (j). Published on Web 08/23/2003 11202 9 J. AM. CHEM. SOC. 2003, 125, 11202-11203 10.1021/ja037262w CCC: $25.00 © 2003 American Chemical Society