Single-Molecule Chiral Recognition on a Surface by Chiral Molecular

Aug 12, 2008 - Japan Science and Technology Agency. , §. Present address: Musashino University, Nishitokyo-shi, Tokyo 202-8585, Japan. Cite this:Anal...
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Anal. Chem. 2008, 80, 6968–6973

Single-Molecule Chiral Recognition on a Surface by Chiral Molecular Tips Tomoaki Nishino†,‡ and Yoshio Umezawa*,†,§ Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan Chiral surfaces attract increasing interest due to their vital role in a variety of scientific fields, such as chiral separation and heterogeneous enantioselective catalysis. The most urgent issue in research on such two-dimensional chirality is a lack of methodologies that recognize molecular chirality on a surface. Here we show that the chiral molecular tips enable for the first time discrimination of enantiomers on a single-molecule basis. The chiral selectivity is attributed to favorable chemical interactions that the molecular tips form with only one of two enantiomers. The stereoselective observation reveals spatial distribution of the enantiomers on a surface at the molecular level. The chiral molecular tips open a way for control of organization of enantiomers toward the advanced functionality of these chiral surfaces through knowledge on pivotal roles of chirality on molecular assemblies as shown here. Modification of solid surfaces by chiral molecules provides important applications, such as in sensors, molecular recognition, and catalysis. The development of the functional chiral interfaces essentially requires establishing fundamental understanding of adsorption structures and intermolecular interactions of chiral molecules on a surface. Recently, some chiral molecules have been reported to form enantiopure domains that result from spontaneous separation of enantiomers upon adsorption onto achiral surfaces.1 The chirality of the segregated two enantiomers is reflected in the mirror-image molecular arrangement in these domains, and many studies convincingly demonstrate such spontaneous enantioseparation by scanning tunneling microscopy (STM) taking advantage of these molecular arrangements.2-8 * To whom correspondence should be addressed. Phone, Fax: +81 42 468 9292. E-mail, [email protected]. † The University of Tokyo. ‡ Japan Science and Technology Agency. § Present address: Musashino University, Nishitokyo-shi, Tokyo 202-8585, Japan. (1) Paci, I.; Szleifer, I.; Ratner, M. A. J. Am. Chem. Soc. 2007, 129, 3545– 3555. (2) Barlow, S. M.; Raval, R. Surf. Sci. Rep. 2003, 50, 201–341. (3) Ku ¨ hnle, A.; Linderoth, T. R.; Hammer, B.; Besenbacher, F. Nature 2002, 415, 891–893. (4) Ku ¨ hnle, A.; Linderoth, T. R.; Besenbacher, F. J. Am. Chem. Soc. 2003, 125, 14680–14681. (5) De Feyter, S.; De Schryver, F. C. Chem. Soc. Rev. 2003, 32, 139–150. (6) Huang, T.; Hu, Z.; Zhao, A.; Wang, H.; Wang, B.; Yang, J.; Hou, J. G. J. Am. Chem. Soc. 2007, 129, 3857–3862. (7) Fang, H.; Giancarlo, L. C.; Flynn, G. W. J. Phys. Chem. 1998, 102, 7311– 7315.

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However, STM is in general silent on molecular chirality itself: enantiomers of both the same and opposite chirality are observed as an identical shape and with identical tunneling current (height or brightness). This is because of limited spatial resolution and chemical selectivity to directly observe and discriminate an asymmetric configuration of a stereogenic carbon atom. Due to such limitations, which is not only in STM but also in almost all the currently available experimental methods, little is known on the molecular level about how a chiral molecule assembles on a surface in the presence of both the enantiomers or how a chiral molecule on a surface interacts with a chiral molecule of a different kind.9 Understanding such behaviors of chiral adsorbates is essential for developing functional chiral surfaces and interfaces including heterogeneous enantioselective catalysis, which is currently a subject of extensive research.10,11 Herein we developed chiral molecular tips in order to achieve chiral recognition of a single molecule. The chiral molecular tips were designed to form favorable chemical interactions with one of enantiomers of a variety of chiral molecules by drawing analogy with chiral recognition in liquid chromatography. As shown in our previous studies,12-14 the chemical interactions provide overlap of electronic wave functions of sample and tip molecules, and the electron tunneling between these molecules is facilitated. For example, we have found the facilitated electron tunneling mediated by hydrogen bonding between a DNA base pair with the use of nucleobase molecular tips. We have demonstrated selective visualization of a particular DNA nucleobase on the basis of the increased electron tunneling.13 Lindsay and co-workers have recently also reported the identification of base-pairing utilizing the molecular tips as above.15 We have found that electron tunneling between the chiral molecular tip and the target enantiomer is much enhanced compared to that between the same chiral tip and the opposite enantiomer. The molecular tips thereby enable stereoselective observation on a single-molecule basis. The (8) Lopinski, G. P.; Moffatt, D. J.; Wayner, D. D. M.; Wolkow, R. A. Nature 1998, 392, 909–911. (9) Chen, Q.; Richardson, N. V. Nat. Mater. 2003, 2, 324–328. (10) Murzin, D. Y.; Maki-Arvela, P.; Toukoniitty, E.; Salmi, T. Catal. Rev.sSci. Eng. 2005, 47, 175–256. (11) Bonello, J. M.; Williams, F. J.; Lambert, R. M. J. Am. Chem. Soc. 2003, 125, 2723–2729. (12) Nishino, T.; Ito, T.; Umezawa, Y. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5659–5662. (13) Ohshiro, T.; Umezawa, Y. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 10– 14. (14) Nishino, T.; Ohshiro, T.; Umezawa, Y. Jpn. J. Appl. Phys. 2007, 46, 5519– 5527. (15) He, J.; Lin, L.; Zhang, P.; Lindsay, S. Nano Lett. 2007, 7, 3854–3858. 10.1021/ac800818f CCC: $40.75  2008 American Chemical Society Published on Web 08/12/2008

Figure 1. Structures of molecules capable of chiral recognition, along with chemical structures of the sample. (a) Schematic of the chiral molecular tips. (b) Precursor molecules of (R)- and (S)-chiral molecular tips (top and bottom, respectively). (c) Chiral selector from which a chiral stationary phase is derived. DNP and n-Pr stand for 3,5dinitrophenyl and n-propyl, respectively. (d) Sample cysteine and (e) 3-mercaptopropionic acid. Stereogenic centers are indicated with asterisks.

facilitation is ascribed to the chemical interactions that chiral molecular tip enantioselectively forms with one of the enantiomers. The stereoselective observation demonstrates that asymmetric interaction between chiral adsorbates significantly affects their surface assemblies. EXPERIMENTAL SECTION General. All reagents were of the highest grade available and used as received. De-ionized water purified with a Milli-Q water purification system (Japan Millipore, Tokyo, Japan) was used throughout the experiments. STM observation was performed on a Nanoscope E (Digital Instrument, Santa Barbara, CA). Synthesis of Tip Molecules. (S)- and (R)-N-(3,5-dinitrobenzoyl)cysteine n-propylamide (Figure 1b) were synthesized by amidations of (S)-trityl-protected D- and L-cysteine, respectively, following the procedure employed for the preparation of a chiral stationary phase (CSP, see Results and Discussion) shown in Figure 1c.16 The trityl group was deprotected under a mild acidic condition (2% trifluoroacetic acid in CH2Cl2) to preserve the stereochemistry of the tip molecules.17 Preparation of Chiral Molecular Tips. Au STM tips were prepared by electrochemical etching of small pieces of Au wire (0.25-mm diameter, Nilaco Co., Tokyo, Japan; 99.95%) in 3 M NaCl at ac 10 V. They were washed by sonicating in pure water and further dipping in “piranha solution” (7:3 H2SO4/H2O2. Caution: piranha solution reacts violently with organic compounds and should not be stored in closed containers) and finally washed thoroughly (16) Pirkle, W. H.; Pochapsky, T. C. J. Am. Chem. Soc. 1987, 109, 5975–5982. (17) Moreau, X.; Campagne, J.-M. J. Org. Chem. 2003, 68, 5346–5350.

with pure water. The chiral molecular tips (Figure 1a) were constructed by self-assembly of the tip molecules (Figure 1b) onto underlying Au tips. For the formation of the self-assembled monolayer, the tip molecules was dissolved in ethanol at a typical concentration of 5 mM, and the gold tips were immersed in the solution overnight. The modified tips were successively rinsed with ethanol and pure water prior to use. Sample Preparation and STM Observation. A gold singlecrystal bead was prepared by melting an end of a gold wire in a hydrogen-oxygen flame. The (111) facet on the bead (see Supporting Information Figure S-1) was employed as a substrate for cysteine self-assembled monolayers (SAMs). The SAMs were formed by immersing the Au bead into an aqueous solution containing either D-, L-, or racemic cysteine (2.5 mM) overnight. The SAM of 3-mercaptopropionic acid (MPA) was similarly prepared by dipping the Au bead into a 2.5 mM ethanolic solution of MPA overnight. The substrate was subsequently washed with pure water to remove physisorbed excess cysteine molecules. The resultant SAMs were observed with STM under ambient conditions at room temperature. Typical bias voltages were in the range of +50 to +450 mV or -50 to -450 mV and the tunneling current was 0.65-1.00 nA. The molecular resolution was hardly achieved outside these ranges with both the molecular and metal tips. Neither of the parameters affects the image contrast selective to the molecular chirality. The stereoselective observation was achieved with ∼30-40% of the chiral molecular tips, and the others exhibited the same STM images as those observed with unmodified metal tips. This is most probably caused by the absence of the tip molecules at the very apex of the underlying gold tip at the atomic level. RESULTS AND DISCUSSION First, the chiral molecular tips were used to observe SAMs of enantiopure cysteine (Figure 1d) on Au(111) under ambient conditions. The cysteine adlayers have been extensively studied3,4,18-23 by a variety of techniques including conventional STM aiming at introducing sophisticated functionalities toward realization of bioinorganic hybrid materials by conjugation of peptide thereon. With the (S)-chiral molecular tips (Figure 1b, bottom), STM images as shown in Figure 2a and b were observed (see also Supporting Information Figure S-2 for additional STM images). It should be noted that the two images significantly differ in the vertical ranges (see below). The images exhibit closely packed arrays of the cysteine molecules adsorbed on the surface. The earlier studies established that the cysteine molecules, as well as alkanethiols with straight hydrocarbon chains,24 form a densely packed ordered adlayer designated as a (3 × 3)R30° structure.21 The molecular packing within the SAMs observed with the (S)-chiral molecular tip (Figure 2) as well as those observed (18) Di Felice, R.; Selloni, A. J. Chem. Phys. 2004, 120, 4906–4914. (19) Nazmutdinov, R. R.; Zhang, J.; Zinkicheva, T. T.; Manyurov, I. R.; Ulstrup, J. Langmuir 2006, 22, 7556–7567. (20) Xu, Q.-M.; Wan, L.-J.; Wang, C.; Bai, C.-L.; Wang, Z.-Y.; Nozawa, T. Langmuir 2001, 17, 6203–6206. (21) Ku ¨ hnle, A.; Linderoth, T. R.; Schunack, M.; Besenbacher, F. Langmuir 2006, 22, 2156–2160. (22) Zhang, J.; Chi, Q.; Nielsen, J. U.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Langmuir 2000, 16, 7229–7237. (23) Graff, M.; Bukowska, J. J. Phys. Chem. B 2005, 109, 9567–9574. (24) Ulman, A. Chem. Rev. 1996, 96, 1533–1554.

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Figure 2. STM observation of cysteine SAMs with the (S)-chiral molecular tip. STM images of (a) D-cysteine and (b) L-cysteine. Bias voltages, -200 mV; tunneling currents, 0.75 nA; scale bars, 2.0 nm. (c) Cross-sectional profiles of the cysteine enantiomers taken with the (S)-chiral molecular tips.

with the (R)-chiral molecular tip (Figure 1b, top), is consistent with these previous reports although other structures were reported under aqueous environment19,20,22 or on a Au(110) surface.3,4 These consistent results demonstrate that the chiral molecular tips probe the electron tunneling from individual sample molecules and reveal their adsorbed structures at the molecular level. The SAM structures of both the D- and L-cysteine enantiomers are identical to each other, and as a result, the D- and L-cysteine molecules are indistinguishable from their molecular packing in the STM images. However, the most important observation is the extent of intermolecular electron tunneling between the chiral molecular tips and cysteine enantiomers. Figure 2c shows cross-sectional profiles of the STM images of the cysteine enantiomers taken with the (S)-chiral molecular tips (see Supporting Information Figure S-3 for the profiles observed with (R)-chiral molecular tips and with metal tips). The profiles do not represent simple physical height in STM because the tunneling current reflects not only topography but also electronic structure of an analyte molecule. With molecular tips, profiles are a quantitative measure of intermolecular electron tunneling between the sample and tip molecules. As the chemical interaction between the sample and tip facilitates electron tunneling in-between, the sample molecules are observed as higher (brighter) protrusions in the images under a constant-current mode. With the (S)-chiral molecular tip, the height of the L-cysteine was found to be 2.8 ± 0.4 Å, while that of the D-cysteine was 0.90 ± 0.24 Å (Figure 3a, middle). This demonstrates that the (S)-chiral molecular tip enhanced electron tunneling from L-cysteine molecules to a much larger extent than from D-cysteine molecules. Interestingly, the oppositely handed 6970

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Figure 3. Chiral recognition via an extent of intermolecular electron tunneling. (a) Bar graphs showing extent of the intermolecular electron tunneling from the cysteine molecules in the enantiopure SAMs to the metal tip (left), (S)-chiral molecular tip (middle), and (R)-chiral molecular tip (right). The bars labeled with “L” and “D” illustrate the extent of the tunneling for L- and D-cysteine, respectively. (b) Bar graphs showing the extent of the intermolecular electron tunneling from the cysteine molecules in the racemic SAMs to the (S)- and (R)-chiral molecular tips (left and right, respectively). In each panel, the left and right bars illustrate the extent of the tunneling for the cysteine molecules appeared as brighter and dimmer protrusions in the STM images, respectively. (c) Bar graphs showing the extent of the intermolecular electron tunneling from MPA molecules to the metal tip (left), (S)-chiral molecular tip (middle), and (R)-chiral molecular tip (right).

molecular tip, (R)-chiral molecular tip, caused a reversed situation, i.e., a larger tunneling current from D-cysteine than from L-cysteine (2.2 ± 0.2 and 0.89 ± 0.14 Å, respectively; Figure 3a, right). In contrast, metal tips exhibited the same tunneling current from both the D- and L-cysteine molecules (0.47 ± 0.06 and 0.37 ± 0.06 Å, respectively; Figure 3a, left), showing that the conventional metal tips are completely silent on the chirality. The chiral molecular tips were also used to observe SAMs of MPA. MPA (Figure 1e) lacks an NH2 group as compared to cysteine (Figure 1d) and, as a result, is not chiral. The two chiral molecular tips exhibited the same tunneling current (Figure 3c, middle and right), which is larger than the current measured with a metal tip (Figure 3c, left) due to hydrogen bonding between the molecular tips and MPA, within experimental error for the MPA molecules. This observation suggests that the stereochemistry of the chiral molecular tips alone does not cause any difference in the extent of the tunneling current as far as a sample molecule is identical. Taken together, these results illustrate that the chiral molecular tips discriminate the stereochemistry of the cysteine molecules by the differing extent of the intermolecular electron tunneling. The chiral molecular tips (Figure 1a) were designed according to a CSP derived from a molecule shown in Figure 1c in highperformance liquid chromatography (HPLC).25,26 The CSP tethered to a solid support is able to separate enantiomers of numerous kinds of chiral molecules as a result of a set of stereoselective interactions formed preferentially between the CSP and one of enantiomers. Chiral recognition of N-(2-naphthyl)alanine ester by the CSP prepared with the molecule in Figure 1c, for instance, results from two hydrogen bonds in addition to a charge-transfer interaction between the aromatic moieties of the (25) Ahuja, S. Chiral Separations by Chromatography; American Chemical Society: Washington, DC, 2000. (26) Pirkle, W. H.; Pochapsky, T. C. Chem. Rev. 1989, 89, 347–362.

CSP and analyte.16 One of the hydrogen bonds is formed between the carbonyl of the CSP and N-terminal amino proton of the sample aniline derivative, and the other between the amide proton of the CSP and C-terminal carbonyl of the analyte. In the chiral molecular tips, the bulky tert-butyl group in the CSP was replaced with a CH2SH substituent (Figure 1b), and its SH group is utilized to immobilize the tip molecule onto also the bulky Au STM tip by S/Au bonding. Because all the other functional groups important for chiral recognition were left unchanged, the chiral molecular tips achieve stereoselective observation as seen in Figure 3a analogously to the CSP. The chiral molecular tip forms favorable hydrogen bond interactions preferentially with one of the sample cysteine enantiomers. Similar to the case of the CSP, the carbonyl and amide proton of the chiral molecular tips enantioselectively form two hydrogen bonds with N-terminal amino proton and C-terminal carbonyl of the cysteine molecule, respectively. The enantioselective formation of the chemical interactions causes the facilitation of the intermolecular electron tunneling from the one enantiomer to a greater extent than the opposite enantiomer (see Supporting Information Figures S-4 and S-5 for the illustration and additional discussion of the enantioselective interactions), whereas these interactions result in longer retention times for a particular enantiomer in HPLC with the CSP. The reversal of the stereoselectivity by replacing the (S)-chiral molecular tip with the (R)-isomer is clear evidence for the enantioselective formation of the hydrogen bond interactions. Furthermore, chemical force microscopy (CFM) employing similarly functionalized tips substantiates such interactions between chiral molecules on tip and substrate.27 Tips modified with molecules similar to those shown in Figure 1c were used to measure adhesion forces with SAMs of a chiral mandelic acid derivative, and the intermolecular forces were found to depend on the chiralities of the sample and tip molecules. It should be emphasized that single-molecule chirality is successfully recognized in the present work (Figure 3a). In contrast, CFM could differentiate 2-µm-wide striped regions where each of the two enantiomers exclusively exist in an alternative way.27 As a result of the outstanding spatial resolution for chirality, the present chiral molecular tips bring insight into pivotal roles of chirality in molecular assemblies on the molecular level (see below). In addition, the chiral molecular tips were found to achieve atomic resolution of highly oriented pyrolytic graphite (see Supporting Information Figure S-6, inset). They naturally exhibited the same images as those obtained with a metal tip in this particular case due to no chemical interaction between the molecular tip and sample. The clear spatial resolution is an indication that a single tip molecule exists at the apex of an underlying Au tip as discussed in our previous paper12 because a tip terminated with multiple protrusions has been known to exhibit strongly distorted images.28 We therefore conclude that the single tip molecule at the tip extremity interacts with a sample molecule and that the chiralselective imaging is achieved on a single-molecule basis. The sample cysteine molecules in the present study lack aromatic moieties for the formation of the charge-transfer interactions, which are sometimes involved to ease the enantioseparation by CSPs. However, the charge-transfer interactions are not a

prerequisite as evidenced by successful chiral recognition by hydrogen bond interactions alone in HPLC.26,29 On the other hand, we preserved an aromatic 3,5-dinitrophenyl moiety in the chiral molecular tips to keep close analogy with the CSP (see below). Some of electroactive moieties, including a nitrophenyl group, have been known to exhibit nonlinear current-voltage (I-V) properties, such as negative differential resistance (NDR).30 The I-V curves were measured with the chiral molecular tips (Supporting Information Figure S-6), and no NDR behavior was observed within the voltage range of -1.0 to 1.0 V. This result rules out the possibility that the electroactive property of the molecular tip plays a role in the intermolecular electron tunneling between sample and tip molecules. It is noteworthy that numerous kinds of chiral analyte are separable on the CSP (Figure 1c).26 We expect that the chiral molecular tips inherit such wide applicability for stereoselective observation of a variety of chiral molecules on surfaces because of the similar mechanism for chiral recognition discussed above in addition to the structural relevance between the chiral molecular tip and CSP. The molecular tips and sample surfaces were prepared by selfassembly of the aromatic or alkanethiols (Figure 1b, d, and e). SAMs consisting of such organosulfur compounds are suitable to create surfaces free of contamination, e.g., adventitious carboneous materials frequently found on an unfunctionalized gold surface. But there exists an adsorbed water layer on the SAM surfaces under ambient conditions. One might expect that the water molecules affect the intermolecular electron tunneling between the tip and sample molecules because water can disrupt the hydrogen bonding in-between, which enhances the electron tunneling. Against this assumption, recent study has shown that the tunneling current through hydrogen bond interactions is not much affected by the presence of water.15 In the present study, the tunneling current, measured under ambient conditions, is much facilitated when the molecular tip forms the enantioselective hydrogen bond interactions with a particular sample enantiomer. Furthermore, the extent of the electron tunneling is reproducible. The relative standard deviation of the extent of the electron tunneling (or apparent height) was 17.3% for the chiral molecular tips (Figure 3a, middle and right panels), and this is comparable to the corresponding value for the metal tip (15.1%; Figure 3a, left panel). These results exclude the possibility of the unwanted effect of the atmospheric water adsorbed on the SAM surfaces of the tip or sample, being consistent with the literature.15 While the molecular arrangements of the enantiopure cysteine SAMs have been extensively investigated both experimentally and theoretically,18-22 little is known about those of a racemic cysteine monolayer,23 in which the two enantiomers coexist. The chiral molecular tips were then applied to observation of the racemic cysteine adlayer in order to investigate how the chirality affects the molecular organization on a surface. Surprisingly, the adsorbates arrange themselves randomly, and the ordered structure as seen in Figure 2 is lost once the two enantiomers are adsorbed together (Figure 4). The racemic monolayers were also prepared at elevated temperature (70 °C) and for prolonged time for the adsorption (24 h). But no improvement in the ordering of the

(27) McKendry, R.; Theoclitou, M.-E.; Rayment, T.; Abell, C. Nature 1998, 391, 566–568. (28) Park, S.-I.; Nogami, J.; Quate, C. F. Phys. Rev. B 1987, 36, 2863–2866.

(29) Allenmark, S.; Schurig, V. J. Mater. Chem. 1997, 7, 1955–1963. (30) Carroll, R. L.; Gorman, C. B. Angew. Chem., Int. Ed. 2002, 41, 4378–4400.

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Figure 4. Stereoselective observation of racemic cysteine monolayer. STM images observed with the (a) (S)-chiral molecular tip, (b) (R)-chiral molecular tip, and (c) metal tip. ∆z scale range, (a, b) 1.5, (c) 0.35, and 0.15 nm (inset). The red circle in (b) indicates a region where D-isomers of cysteine exclusively exist. Bias voltages, -200 mV; scale bars, 3.0 nm. Tunneling currents; (a) 0.75, (b) 0.65, and (c) 1.0 nA. (d) A cross-sectional profile of the cysteine enantiomers measured in (a). The arrows labeled with “D” and “L” indicate the position of the D- and L-isomers, respectively.

adsorbate arrangement was found, indicating that the disordered structure is in, or at least near, thermodynamic equilibrium. The metal tips can image the randomly distributed each adsorbate (Figure 4c). Unfortunately, however, they are completely insensitive to molecular chirality (Figure 3a, left) and, as a result, fail to explore the stereochemistry within the racemic adlayer. By contrast, the (S)-chiral molecular tip exhibited markedly different image contrasts in the STM images (Figure 4a). It should be noted that the z-scale ranges of the images in Figure 4 are not the same, and the image observed with a metal tip (Figure 4c) is much flatter than the images observed with the chiral molecular tips (Figure 4a and b). The disordered structure was found in Figure 4a, and this is consistent with the observation using the metal tips. More importantly, two kinds of image contrast or height, which represents an extent of electron tunneling, are discerned with the chiral molecular tip, and the cysteine molecules appear as either brighter or dimmer protrusions in Figure 4a (Figure 4d). The heights of these protrusions were 2.8 ± 0.4 and 1.3 ± 0.2 Å for the brighter and dimmer ones, respectively (Figure 3b, left). Through the observation of enantiopure cysteine SAMs described above, we have found that the (S)-chiral molecular tip selectively visualizes the L-cysteine molecule as brighter protrusions than the D-isomer (Figure 3a, middle). Moreover, the heights of the brighter and dimmer protrusions agree well with those of L- and D-cysteine, respectively. On the basis of these results, the brighter molecules in the images observed with the (S)-chiral molecular tip (Figure 4a) are unequivocally assigned as the L-cysteine molecules. Similarly, with the (R)-chiral molecular tip the adsorbed cysteine molecules exhibited brighter (2.5 ± 0.4 Å) and dimmer (1.3 ± 0.2 Å) contrast (Figure 4b and Figure 3b, right). These values are again well consistent with the heights of D- and L-cysteine measured with the (R)-chiral molecular tips (Figure 3a, right). It is therefore concluded that the brighter molecules correspond to D-cysteine in the case of the (R)-chiral molecular 6972

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Figure 5. Models of enantiopure cysteine SAMs. Au(111) surface atoms were omitted for the sake of clarity. The black rhombuses represent unit cells of the monolayers. Blue and red circles indicate an amino group and oxygen-containing functional groups, respectively, which are involved in intermolecular hydrogen bond interactions.

tip. As seen in Figure 4a and b, the single-molecule stereoselective observation achieved by the chiral molecular tips revealed that the two enantiomers intermix together within the disordered racemic monolayer. Figure 5 shows models of adlayer structures consisting of enantiopure cysteine. For the construction of the models, the orientation of the cysteine molecules was based on theoretical calculations,18,19 and these cysteine molecules were arranged following the structure observed in this work (Figure 2) as well as in the literature.21 These models clearly show that the enantiomers form mirror-image monolayers. This chirality of the monolayer arises due to asymmetric configurations of the amino and carboxy groups of the cysteine molecules (blue and red circles in Figure 5, respectively). It should be noted, however, that the arrangements of the whole molecules (black rhombuses in Figure 5) are superimposable by rotation and identical to each other. The amino and carboxy groups of the cysteine molecule cannot be resolved in the STM images, and each molecule is observed as a single protrusion. This limited spatial resolution makes the chirality of the monolayer invisible in STM observation using conventional metal tips. In general, interactions between adsorbates as well as those between an adsorbate and substrate are decisive factors in an adlayer structure. In the present cysteine monolayers, hydrogen bond interactions involving the carboxy and amino groups play central roles in this context. The structural models in Figure 5 indicate that there exists a hydrogen bond network involving the carboxy and amino groups between the homochiral cysteine molecules in the well-ordered enantiopure SAMs. In the case of

the racemic monolayer, the cysteine molecules are surrounded by both of the enantiomers, and this situation prevents formation of the hydrogen bond network and consequently causes the disordered structure as seen in Figure 4. In fact, local ordering was observed at the area where the cysteine molecules of the same chirality exclusively adsorb next to one another. Figure 4b shows an example of such behavior. In the region marked with the red circle, tens of D-cysteine molecules, observed as bright protrusions with the (R)-chiral molecular tip, form a locally ordered structure as seen in the enantiopure SAMs. If a cysteine SAM contains a higher amount of one enantiomer than the other, the abundant enantiomer has more chance to interact with the same kind of enantiomer. This results in frequent formation of the homochial interaction, and as a result, the surface ordering would be increased under the enantiomeric excess condition. The difference in the intermolecular hydrogen bond interactions between enantiopure and racemic SAMs agrees with recent observation with surface-enhanced Raman scattering spectroscopy (SERS). The SERS study of the cysteine SAMs reveals considerable lowering of a Raman frequency of the carboxy-related band, which is sensitive to the interaction, for racemic cysteine as compared to enantiopure cysteines.23 CONCLUSIONS In summary, chiral molecular tips were developed for chiral recognition of a single molecule on a surface. The stereoselectivity stems from enantioselective interactions of the molecular tip with a sample molecule of a particular chirality. The chiral molecular

tips successfully discriminate each of the intermixed enantiomers in the racemic monolayer of cysteine. These results illustrate a pivotal role of the enantiomeric interactions in the molecular organization on a surface. We believe that the chiral molecular tips pave the way for research on single-molecule stereochemistry. ACKNOWLEDGMENT This work is supported by a grant from the Japan Science and Technology Agency (JST), Grants for Scientific Research from the Ministry of Education, Science and Culture, Japan, and partly by Sasakawa Scientific Research Grant from The Japan Science Society. SUPPORTING INFORMATION AVAILABLE An STM images of a flat Au(111) terrace (Figure S-1), additional STM images of cysteine SAMs observed with (S)-chiral molecular tip (Figure S-2), cross-sectional profiles of the STM images of enantiopure cysteine SAMs observed with the (S)-, (R)chiral molecular tips and metal tip (Figure S-3), illustrations of the enantioselective interactions between the chiral molecular tips and cysteines (Figures S-4 and S-5), and current-voltage curves measured with the chiral molecular tips (Figure S-6). This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 23, 2008. Accepted July 17, 2008. AC800818F

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